Therapeutic use of antibodies against ENPP3

ABSTRACT

Disclosed herein are antibodies against ENPP3 and uses thereof, specifically monoclonal antibodies against ENPP3, bispecific antibodies against ENPP3 and CD3, nucleic acids including nucleotide sequences encoding the antibodies, vectors including the nucleic acids, and host cell including the nucleic acids or the vectors. Also disclosed are pharmaceutical compositions and conjugates including the antibodies, and therapeutic methods for using the antibodies.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of International Patent ApplicationNo. PCT/CN2022/097597, filed on Jun. 8, 2022. For all purposes under thelaw, the entire disclosure of the aforementioned application isincorporated by reference as part of the disclosure of this application.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing(L047370041US00-SUBSEQ-DFC.xml; Size: 46,779 bytes; and Date ofCreation: Apr. 3, 2023) is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention is directed to antibodies against ENPP3, and usesof such antibodies, in particular their use in the treatment of cancers.

BACKGROUND OF THE INVENTION

Ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3, also known asCD203c), a 150 kDa protein, belongs to a series of ectoenzymes thathydrolyze extracellular pyrophosphate or phosphodiester bonds as well asintracellular nucleotides involved in glycosyltransferase activity.These ectoenzymes possess ATPase and ATP pyrophosphatase activities andare type II transmembrane proteins.

Previous studies indicated ENPP3 has functional relevance in multiplebiological processes, for instance endometrial receptivity and embryoimplantation. ENPP3 is upregulated in neoplastic mast cells in thecontext of mastocytosis, in acute basophilic leukemia, in neoplasticcells of the bile duct. More notably, ENPP3 has been found to play animportant role in the development and invasion of tumors.

ENPP3 is expressed in multiple organs and cell types, includingepithelial, mucosal surfaces, and notably basophils and mast cells. Incontrast to the restricted expression in normal tissues, ENPP3 is highlyexpressed in renal cell carcinoma (RCC) and subsets of hepatocellularcarcinoma (HCC). The expression pattern of ENPP3 shows the potentialcapability for tumor therapy.

SUMMARY OF THE INVENTION

The present disclosure provides novel antibodies targeting ENPP3 orantigen binding fragments thereof, which can be in a form of amonoclonal antibody or bispecific antibody, such as a bispecific T-cellengager (BiTE®). A variety of functional assays have demonstrated thepotent anti-tumor effect of the antibodies.

In an aspect, the present disclosure provides an antibody specificallybinding to ENPP3, or an antigen binding fragment thereof, comprising alight chain variable region (VL) and a heavy chain variable region (VH),wherein the VL comprises LCDRs 1-3 having the amino acid sequences asset forth in SEQ ID NOs: 1-3 respectively, and the VH comprises HCDRs1-3 having the amino acid sequences as set forth in SEQ ID NOs: 6-8respectively.

In some embodiments of the antibody or the antigen binding fragmentthereof disclosed herein, the VL comprises an amino acid sequence havingat least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100% sequence identity to SEQ ID NO: 4, and the VHcomprises an amino acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100% sequenceidentity to SEQ ID NO: 9. In some embodiments, the VL comprises an aminoacid sequence as set forth in SEQ ID NO: 4 and the VH comprises an aminoacid sequence as set forth in SEQ ID NO: 9.

In some embodiments, the antibody is of an isotype selected from thegroup consisting of IgG, IgA, IgM, IgE and IgD. In some embodiments, theantibody is of a subtype selected from the group consisting of IgG1,IgG2, IgG3, and IgG4.

In some embodiments, the antigen binding fragment can be selected fromthe group consisting of Fab, Fab′, F(ab′)₂, Fv, scFv, and ds-scFv.

In some embodiments, the antibody can be a monoclonal antibody. In someembodiments, the antibody comprises a light chain comprising an aminoacid sequence having at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:5 and a heavy chain comprising an amino acid sequence having at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100% sequence identity to SEQ ID NO: 10.

In other embodiments, the antibody can be a bispecific or amulti-specific antibody. In some embodiments, the antibody can be abispecific antibody which further comprises a second antigen bindingregion binding to a second antigen. In some embodiments, the secondantigen can be a tumor associated antigen or an immune cell antigen. Insome embodiments, the second antigen can be a T-cell antigen. In someembodiments, the T-cell antigen can be selected from the groupconsisting of T cell receptor (TCR), CD3, CD4, CD8, CD16, CD25, CD28,CD38, CD44, CD62L, CD69, ICOS, 41-BB (CD137), and NKG2D.

In some embodiments, the second antigen is CD3, and the second antigenbinding region comprises a VL and a VH, wherein the VL comprises LCDRs1-3 having the amino acid sequences as set forth in SEQ ID NOs: 11-13respectively, and the VH comprises HCDRs 1-3 having the amino acidsequences as set forth in SEQ ID NOs: 16-18 respectively.

In some embodiments, the second antigen binding region comprises a VLcomprising an amino acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100% sequenceidentity to SEQ ID NO: 14 and a VH comprising an amino acid sequencehaving at least 80%, at least 85%, at least 90%, at least 95%, at least98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19. In someembodiments, the second antigen binding region comprises a VL comprisingan amino acid sequence as set forth in SEQ ID NO: 14 and a VH comprisingan amino acid sequence as set forth in SEQ ID NO: 19.

In some embodiments, the VL of the second antigen binding region islinked to the C-terminal of the VL of the antibody specifically bindingto ENPP3, optionally via a first linker, and the VH of the secondantigen binding region is linked to the C-terminal of the VH of theantibody specifically binding to ENPP3, optionally via a second linker,wherein the first linker and the second linker are the same ordifferent.

In some embodiments, each of the first linker and the second linkerindependently comprises an amino acid selected from SEQ ID NO: 21(GSGGGGSGGGGS) and SEQ ID NO: 22 (GSGGSGGGGSGGGGS). In some embodiments,the first linker comprises an amino acid sequence as set forth in SEQ IDNO: 21 (GSGGGGSGGGGS), and the second linker comprises an amino acidsequence as set forth in SEQ ID NO: 22 (GSGGSGGGGSGGGGS).

In some embodiments, the bispecific antibody comprises a light chaincomprising an amino acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100% sequenceidentity to SEQ ID NO: 15 and a heavy chain comprising an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 20.

In some embodiments, the bispecific antibody is a bispecific T-cellengager (BITE®).

In another aspect, the present disclosure provides a bispecific antibodyor an antigen binding fragment thereof, comprising a first antigenbinding region binding to ENPP3 comprising a VL and a VH and a secondantigen binding region binding to CD3 comprising a VL and a VH, whereinthe VL of the first antigen binding region comprises LCDRs 1-3 havingthe amino acid sequences as set forth in SEQ ID NOs: 1-3 respectively,and the VH of the first antigen binding region comprises HCDRs 1-3having the amino acid sequences as set forth in SEQ ID NOs: 6-8respectively; and the VL of the second antigen binding region comprisesLCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs:11-13 respectively, and the VH of the second antigen binding regioncomprises HCDRs 1-3 having the amino acid sequences as set forth in SEQID NOs: 16-18 respectively.

In some embodiments of the bispecific antibody or the antigen bindingfragment thereof disclosed herein, the VL of the first antigen bindingregion comprises an amino acid sequence having at least 80%, at least85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 4 and the VH of the first antigenbinding region comprises an amino acid sequence having at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100% sequence identity to SEQ ID NO: 9; and the VL of the second antigenbinding region comprises an amino acid sequence having at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100% sequence identity to SEQ ID NO: 14 and the VH of the second antigenbinding region comprises an amino acid sequence having at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100% sequence identity to SEQ ID NO: 19.

In some embodiments, the VL of the first antigen binding regioncomprises an amino acid sequence as set forth in SEQ ID NO: 4 and the VHof the first antigen binding region comprises an amino acid sequence asset forth in SEQ ID NO: 9; and the VL of the second antigen bindingregion comprises an amino acid sequence as set forth in SEQ ID NO: 14and the VH of the second antigen binding region comprises an amino acidsequence as set forth in SEQ ID NO: 19.

In some embodiments, the VL of the second antigen binding region islinked to the C-terminal of the VL of the first antigen binding region,optionally via a first linker, and the VH of the second antigen bindingregion is linked to the C-terminal of the VH of the first antigenbinding region, optionally via a second linker, wherein the first linkerand the second linker are the same or different.

In some embodiments, each of the first linker and the second linkerindependently comprises an amino acid selected from SEQ ID NO: 21(GSGGGGSGGGGS) and SEQ ID NO: 22 (GSGGSGGGGSGGGGS). In some embodiments,the first linker comprises an amino acid sequence as set forth in SEQ IDNO: 21 (GSGGGGSGGGGS), and the second linker comprises an amino acidsequence as set forth in SEQ ID NO: 22 (GSGGSGGGGSGGGGS).

In some embodiments, the bispecific antibody comprises a light chaincomprising an amino acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100% sequenceidentity to SEQ ID NO: 15 and a heavy chain comprising an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 20.

In some embodiments, the bispecific antibody can be a bispecific T-cellengager (BITE®).

In still another aspect, the present disclosure provides a nucleic acidcomprising a nucleotide sequence encoding the antibody or the antigenbinding fragment thereof disclosed herein or the bispecific antibody orthe antigen binding fragment thereof disclosed herein.

In yet another aspect, the present disclosure provides a vectorcomprising the nucleic acid disclosed herein.

In another aspect, the present disclosure provides a host cellcomprising the nucleic acid disclosed herein or the vector disclosedherein.

In still another aspect, the present disclosure provides apharmaceutical composition comprising (i) the antibody or the antigenbinding fragment thereof disclosed herein, or the bispecific antibody orthe antigen binding fragment thereof disclosed herein, and (ii) apharmaceutically acceptable carrier or excipient.

In some embodiments of the pharmaceutical composition disclosed herein,the pharmaceutical composition further comprising a second therapeuticagent. In some embodiments, the second therapeutic agent can be selectedfrom an antibody, a chemotherapeutic agent and a small molecule drug. Insome embodiments, the second therapeutic agent can be selected from aBruton's tyrosine kinase (BTK) inhibitor, a PI3K inhibitor, a HDACinhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1/PD-L1 inhibitor, aLAG3 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor,and glucocorticoid.

In yet another aspect, the present disclosure provides a conjugatecomprising the antibody or the antigen binding fragment thereofdisclosed herein or the bispecific antibody or the antigen bindingfragment thereof disclosed herein, and a chemical moiety conjugatedthereto.

In some embodiments of the conjugate disclosed herein, the chemicalmoiety can be selected from the group consisting of a therapeutic agent,a detectable moiety, and an immune stimulatory molecule.

In another aspect, the present disclosure provides a method of treatinga cancer in a subject comprising administering to the subject aneffective amount of the antibody or the antigen binding fragment thereofdisclosed herein, the bispecific antibody or the antigen bindingfragment thereof disclosed herein, the pharmaceutical compositiondisclosed herein, or the conjugate disclosed herein.

In some embodiments of the method disclosed herein, the cancer is anENPP3 positive cancer. In some embodiments, the cancer can be selectedfrom the group consisting of mastocytosis, leukemia, kidney cancer, lungcancer, gastric cancer, ovarian cancer, breast cancer, pancreaticcancer, colon cancer, colorectal cancer, bile duct cancer, liver cancer,and Wilms tumor.

In some embodiments, the method further comprises administering to thesubject a second therapeutic agent. In some embodiments, the secondtherapeutic agent can be selected from an antibody, a chemotherapeuticagent and a small molecule drug. In some embodiments, the secondtherapeutic agent can be selected from a Bruton's tyrosine kinase (BTK)inhibitor, a PI3K inhibitor, a HDAC inhibitor, an ERK inhibitor, a MAPKinhibitor, a PD-1/PD-L1 inhibitor, a LAG3 inhibitor, a CTLA-4 inhibitor,a TIGIT inhibitor, a TIM3 inhibitor, and glucocorticoid.

In another aspect, the present disclosure provides use of the antibodyor the antigen binding fragment thereof disclosed herein, the bispecificantibody or the antigen binding fragment thereof disclosed herein, thepharmaceutical composition disclosed herein, or the conjugate disclosedherein in the manufacture of a medicament for treating a cancer in asubject. In some embodiments, the cancer is an ENPP3 positive cancer. Insome embodiments, the cancer can be selected from the group consistingof mastocytosis, leukemia, kidney cancer, lung cancer, gastric cancer,ovarian cancer, breast cancer, pancreatic cancer, colon cancer,colorectal cancer, bile duct cancer, liver cancer, and Wilms tumor.

In still another aspect, the present disclosure provides the antibody orthe antigen binding fragment thereof disclosed herein, the bispecificantibody or the antigen binding fragment thereof disclosed herein, thepharmaceutical composition disclosed herein, or the conjugate disclosedherein for use in treating a cancer in a subject. In some embodiments,the cancer is an ENPP3 positive cancer. In some embodiments, the cancercan be selected from the group consisting of mastocytosis, leukemia,kidney cancer, lung cancer, gastric cancer, ovarian cancer, breastcancer, pancreatic cancer, colon cancer, colorectal cancer, bile ductcancer, liver cancer, and Wilms tumor.

BRIEF DESCRIPTION OF THE DRAWINGS

An understanding of the features and advantages of the present inventionwill be obtained by reference to the following detailed description thatsets forth illustrative embodiments, in which the principles of theinvention are utilized, and the accompanying drawings of which:

FIG. 1 shows binding of Fab 3A10 against recombinant human ENPP3 asmeasured by ELISA. BSA is used as negative control.

FIG. 2 shows binding of 3A10 mAb against recombinant human ENPP3 asmeasured by ELISA.

FIG. 3A shows binding of 3A10 mAb against ENPP3 positive cancer cellline SK-Nep-1 as measured by flow cytometry. An IgG4 isotype antibody isused as negative control.

FIG. 3B shows binding of 3A10 mAb against LS174T-ENPP3 cells stablyexpressing ENPP3 as measured by flow cytometry. An IgG4 isotype antibodyis used as negative control.

FIG. 4 shows ADCC killing of 3A10 mAb against HepG2 cells in thepresence of NK cells. An IgG4 isotype antibody is used as negativecontrol.

FIG. 5A shows binding of CMD016 against recombinant human ENPP3 asmeasured by ELISA.

FIG. 5B shows binding of CMD016 against recombinant human CD3 asmeasured by ELISA.

FIG. 6A shows binding of CMD016 against ENPP3 positive cancer cell lineSK-Nep-1 as measured by flow cytometry. An IgG4 isotype antibody is usedas negative control.

FIG. 6B shows binding of CMD016 against CD3 positive Jurkat cells asmeasured by flow cytometry. An IgG4 isotype antibody is used as negativecontrol.

FIG. 7 shows killing of CMD016 against LS174T-ENPP3 cells stablyexpressing ENPP3 in the presence of human PBMCs. ENPP3 negative LS174Tcell line is used as negative control.

FIG. 8 shows concentration of CMD016 in serum from CMD016-treated miceat 24 hr, 48 hr, 72 hr and 96 hr after treatment.

FIG. 9A shows tumor volume in mice treated with 33.3 μg/kg, 100 μg/kgand 300 μg/kg of CMD016. The mice treated with PBS are used as negativecontrol.

FIG. 9B shows body weight of mice treated with 33.3 μg/kg, 100 μg/kg and300 μg/kg of CMD016. The mice treated with PBS are used as negativecontrol.

FIG. 9C shows tumor weight in mice treated with 33.3 μg/kg, 100 μg/kgand 300 μg/kg of CMD016. The mice treated with PBS are used as negativecontrol.

DETAILED DESCRIPTION OF THE INVENTION

The aforementioned features and advantages of the invention as well asadditional features and advantages thereof will be more clearlyunderstood hereafter as a result of a detailed description of thefollowing embodiments when taken in conjunction with the drawings.

The embodiments described herein with reference to drawings areexplanatory, illustrative, and used to generally understand the presentinvention. The embodiments shall not be construed to limit the scope ofthe present invention. The same or similar elements and the elementshaving same or similar functions are denoted by like reference numeralsthroughout the descriptions.

Unless indicated or defined otherwise, all terms used have their usualmeaning in the art, which will be clear to the skilled person. Referenceis for example made to the standard handbooks, such as Leuenberger, H.G. W, Nagel, B. and Klbl, H. eds., “A multilingual glossary ofbiotechnological terms: (IUPAC Recommendations)”, Helvetica Chimica Acta(1995), CH-4010 Basel, Switzerland; Sambrook et al, “Molecular Cloning:A Laboratory Manual” (2nd Ed.), Vols. 1-3, Cold Spring Harbor LaboratoryPress (1989); F. Ausubel et al, eds., “Current protocols in molecularbiology”, Green Publishing and Wiley InterScience, New York (1987);Roitt et al., “Immunology (6th Ed.), Mosby/Elsevier, Edinburgh (2001);and Janeway et al., “Immunobiology” (6th Ed.), Garland SciencePublishing/Churchill Livingstone, New York (2005), as well as thegeneral background art cited above.

As used herein, singular forms “a”, “and,” and “the” include pluralreferents unless the context clearly indicates otherwise. Thus, forexample, reference to “an antibody” includes a plurality of antibodiesand reference to “an antibody” in some embodiments includes multipleantibodies, and so forth.

Unless indicated or defined otherwise, the term “comprise”, andvariations such as “comprises” and “comprising”, should be understood toimply the inclusion of a stated elements or step or group of elements orsteps but not the exclusion of any other element or step or group ofelements or steps.

As used herein, the term “antibody” refers to an immunoglobulin moleculewhich has the ability to specifically bind to a specific antigen. Anantibody often comprises a variable region and a constant region in eachof a heavy chain and a light chain. The variable regions of the heavyand light chains of antibodies contain a binding domain that interactswith an antigen. The constant regions of antibodies may mediate thebinding of the immunoglobulin to host tissues or factors, includingvarious cells of the immune system (such as effector cells) andcomponents of the complement system such as C1q, the first component inthe classical pathway of complement activation. Accordingly, mostantibodies have a heavy chain variable region (VH) and a light chainvariable region (VL) that together form the portion of the antibody thatbinds to the antigen.

A “light chain variable region” (VL) or “heavy chain variable region”(VH) consists of a “framework” region interrupted by three“complementarity determining regions” or “CDRs”. The framework regionsserve to align the CDRs for specific binding to an epitope of anantigen. The CDRs include the amino acid residues of an antibody thatare primarily responsible for antigen binding. From amino-terminus tocarboxyl-terminus, both VL and VH domains comprise the followingframework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, andFR4. CDRs 1, 2, and 3 of a VL domain are also referred to herein,respectively, as LCDR1, LCDR2, and LCDR3; CDRs 1, 2, and 3 of a VHdomain are also referred to herein, respectively, as HCDR1, HCDR2, andHCDR3.

The assignment of amino acids to each VL and VH domain is in accordancewith any conventional definition of CDRs. Conventional definitionsinclude, the Kabat definition (Kabat, Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.,1987 and 1991), the Chothia definition (Chothia & Lesk, J. Mol. Biol.196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); acomposite of Chothia Kabat CDR in which CDR-H1 is a composite of Chothiaand Kabat CDRs; the AbM definition used by Oxford Molecular's antibodymodelling software; and the CONTACT definition of Martin et al. (worldwide web bioinfo.org.uk/abs). Kabat provides a widely used numberingconvention (Kabat numbering system) in which corresponding residuesbetween different heavy chains or between different light chains areassigned the same number. The present disclosure can use CDRs definedaccording to any of these numbering systems, although preferredembodiments use Kabat defined CDRs.

The term “antibody” as used herein should be understood in its broadestmeaning, and includes monoclonal antibodies (including full-lengthmonoclonal antibodies), polyclonal antibodies, antibody fragments, andmulti-specific antibodies containing at least two different antigenbinding regions (e.g., bispecific antibodies). The antibody may containadditional modifications, such as non-naturally occurring amino acids,mutations in Fc regions, and mutations in glycosylation sites.Antibodies also include post-translation modified antibodies, fusionproteins containing the antigenic determinants of the antibody, andimmunoglobulin molecules containing any other modifications to antigenrecognition sites, as long as these antibodies exhibit desiredbiological activity.

As used herein, the term “antigen binding fragment” of an antibodyrefers to one or more fragments of an antibody that retain the abilityto specifically bind to an antigen (e.g., an ENPP3 protein). It has beenshown that the antigen binding function of an antibody can be performedby fragments of a full-length antibody.

Examples of antigen binding fragments encompassed within the term“antigen binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fab′ fragment,which is essentially an Fab with part of the hinge region(see,FUNDAMENTALIMMUNOLOGY (Paul ed., 3.sup.rd ed. 1993); (iv) a Fd fragmentconsisting of the VH and CH1 domains; (v) a Fd′ fragment having VH andCH1 domains and one or more cysteine residues at the C-terminus of theCH1 domain; (vi) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (vii) a dAb fragment (Ward et al., (1989)Nature 341:544-546), which consists of a VH domain; (viii) an isolatedcomplementarity determining region (CDR); and (ix) a nanobody, a heavychain variable region containing a single variable domain and twoconstant domains. Furthermore, although the two domains of the Fvfragment, VL and VH are coded for by separate genes, they can be joined,using recombinant methods, by a synthetic linker that enables them to bemade as a single protein chain in which the VL and VH regions pair toform monovalent molecules (known as single chain Fv (scFv); see e.g.,Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies arealso intended to be encompassed within the term “antigen bindingfragment” of an antibody. Furthermore, the term also includes a “linearantibody” comprising a pair of tandem Fd segments (VH-CH1-VH-CH1), whichforms an antigen binding region together with a complementary lightchain polypeptide, and a modified version of any of the foregoingfragments, which retains antigen binding activity.

These antigen binding fragments can be obtained using conventionaltechniques known to those with skill in the art, and the fragments arescreened for utility in the same manner as are intact antibodies.

As used herein, the term “binding” or “specifically binding” refers to anon-random binding reaction between two molecules, such as between anantibody and its target antigen. The binding specificity of an antibodycan be determined based on affinity and/or avidity. The affinity,represented by the equilibrium constant for the dissociation of anantigen with an antibody (KD), is a measure for the binding strengthbetween an antigenic determinant and an antigen-binding site on theantibody: the lesser the value of the KD, the stronger the bindingstrength between an antigenic determinant and the antibody.Alternatively, the affinity can also be expressed as the affinityconstant (KA), which is 1/KD.

Avidity is the measure of the strength of binding between an antibodyand the pertinent antigen. Avidity is related to both the affinitybetween an antigenic determinant and its antigen binding site on theantibody and the number of pertinent binding sites present on theantibody. Typically, an antibody will bind to an antigen with adissociation constant (KD) of 10⁻⁵ to 10⁻¹² M or less, and preferably10⁻⁷ to 10⁻¹² M or less and more preferably 10⁻⁸ to 10⁻¹² M, and/or witha binding affinity of at least 10⁷ M⁻¹, preferably at least 108 M⁻¹,more preferably at least 109 M⁻¹, such as at least 1012 M⁻¹. Any KDvalue greater than 10⁻⁴ M is generally considered to indicatenon-specific binding. Specifically binding of an antibody to an antigenor antigenic determinant can be determined in any suitable manner known,including, for example, Scatchard analysis and/or competitive bindingassays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) andsandwich competition assays, and the different variants thereof known inthe art.

The term “epitope” refers to a site on an antigen to which an antibodybinds. An epitope can be formed from contiguous amino acids ornoncontiguous amino acids juxtaposed by tertiary folding of one or moreproteins. Epitopes formed from contiguous amino acids (also known aslinear epitopes) are typically retained on exposure to denaturingsolvents whereas epitopes formed by tertiary folding (also known asconformational epitopes) are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation. Theepitope defines the smallest binding site of an antibody and thereforeis the specific target of the antibody or antigen binding fragmentthereof.

As used herein, the term “sequence identity” refers to the extent towhich two sequences (amino acid) have the same residue at the samepositions in an alignment. For example, “an amino acid sequence is X %identical to SEQ ID NO: Y” refers to % identity of the amino acidsequence to SEQ ID NO:Y and is elaborated as X % of residues in theamino acid sequence are identical to the residues of sequence disclosedin SEQ ID NO: Y.

Generally, computer programs are employed for such calculations.Exemplary programs that compare and align pairs of sequences, includeALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988;Pearson, 1990) and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN,or GCG (Devereux et al., 1984).

Also, in determining the degree of sequence identity between two aminoacid sequences, the skilled person may take into account so-called“conservative” amino acid substitutions, which can generally bedescribed as amino acid substitutions in which an amino acid residue isreplaced with another amino acid residue of similar chemical structureand which has little or essentially no influence on the function,activity or other biological properties of the polypeptide. Suchconservative amino acid substitutions are well known in the art, forexample from WO 04/037999, GB-A-2 357 768, WO 98/49185, WO 00/46383 andWO 01/09300; and (preferred) types and/or combinations of suchsubstitutions may be selected on the basis of the pertinent teachingsfrom WO 04/037999 as well as WO 98/49185 and from the further referencescited therein.

Such conservative substitutions preferably are substitutions in whichone amino acid within the following groups (a)-(e) is substituted byanother amino acid residue within the same group: (a) small aliphatic,nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b)polar, negatively charged residues and their (uncharged) amides: Asp,Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg andLys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Val and Cys;and (e) aromatic residues: Phe, Tyr and Trp.

Particularly preferred conservative substitutions are as follows: Alainto Gly or into Ser; Arg into Lys; Asn into Gln or into His; Asp intoGlu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro;His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or intoVal; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or intoIle; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trpinto Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into Leu.

Any amino acid substitutions applied to the polypeptides describedherein may also be based on the analysis of the frequencies of aminoacid variations between homologous proteins of different speciesdeveloped by Schulz et al., Principles of Protein Structure,Springer-Verlag, 1978, on the analyses of structure forming potentialsdeveloped by Chou and Fasman, Biochemistry 13: 211, 1974 and Adv.Enzymol., 47: 45-149, 1978, and on the analysis of hydrophobicitypatterns in proteins developed by Eisenberg et al., Proc. Nat. Acad Sci.USA 81: 140-144, 1984; Kyte & Doolittle, J Mol. Biol. 157: 105-132, 1981, and Goldman et al., Ann. Rev. Biophys. Chem. 15: 321-353, 1986, allincorporated herein in their entirety by reference.

As used herein, the term “monoclonal antibody” refers to an antibodyobtained from a substantially homogeneous antibody population. That is,each antibodies constituting the population are the same, except forpossible naturally occurring mutations in small amount. Monoclonalantibodies are highly specific and are directed against a singleantigen. The term “monoclonal antibody” herein is not limited toantibodies produced by hybridoma technology, and should not beinterpreted as requiring production of antibodies by any specificmethod.

The term “bispecific antibody” is in the context of the presentinvention to be understood as an antibody having two differentantigen-binding regions defined by different antibody sequences. Thiscan be understood as different target binding but includes as wellbinding to different epitopes in one target.

As used herein, the term “tumor associated antigen” refers to an antigenthat is differentially expressed in cancer cells compared to normalcells, and therefore can be used to target cancer cells.

As used herein, the term “CD3” refers to the human CD3 protein complex,which has five peptide chains, γ chain, δ chain, ε chain, ζ chain and ηchain, and is associated with the T cell receptor α and β chains to forma TCR-CD3 complex. The term includes any CD3 variants, isoforms andspecies homologs which are naturally expressed by cells, including Tcells, or are expressed on cells transfected with genes or cDNA encodingthe aforementioned chains.

As used herein, the term “bispecific T-cell engager” or “BiTE®” refersto a polypeptide chain molecule having two antigen-binding domains, oneof which binds to a T-cell antigen and the second of which binds to anantigen present on the surface of target cells (See, PCT Publication WO05/061547; Baeuerle et al., 2008, Drugs of the Future 33: 137-147;Bargou, et al., 2008, Science 321:974-977, which are incorporated hereinby reference in their entireties). Thus, the BiTE® of the disclosure hasan antigen binding region that binds to ENPP3 and a second antigenbinding region that is directed towards a T-cell antigen.

As used herein, the term “vector” is intended to refer to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked.

As used herein, the term “host cell” refers to a cell into which anexpression vector has been introduced.

The term “pharmaceutically acceptable” means that the carrier oradjuvant is compatible with the other ingredients of the composition andnot substantially deleterious to the recipient thereof and/or that suchcarrier or adjuvant is approved or approvable for inclusion in apharmaceutical composition for parenteral administration to humans.

As used herein, the terms “treatment”, “treating”, “treat”, and thelike, refer to administering an agent, or carrying out a procedure, forthe purposes of obtaining an effect. The effect may be prophylactic interms of completely or partially preventing a disease or symptom thereofand/or may be therapeutic in terms of effecting a partial or completecure for a disease and/or symptoms of the disease. “Treatment”, as usedherein, may include treatment of a disease or disorder (e.g. cancer) ina mammal, particularly in a human, and includes: (a) preventing thedisease or a symptom of a disease from occurring in a subject which maybe predisposed to the disease but has not yet been diagnosed as havingit (e.g., including diseases that may be associated with or caused by aprimary disease); (b) inhibiting the disease, i.e., arresting itsdevelopment; and (c) relieving the disease, i.e., causing regression ofthe disease. Treating may refer to any indicia of success in thetreatment or amelioration or prevention of a cancer, including anyobjective or subjective parameter such as abatement; remission;diminishing of symptoms or making the disease condition more tolerableto the patient; slowing in the rate of degeneration or decline; ormaking the final point of degeneration less debilitating. The treatmentor amelioration of symptoms is based on one or more objective orsubjective parameters; including the results of an examination by aphysician. Accordingly, the term “treating” includes the administrationof the antibodies or compositions or conjugates disclosed herein toprevent or delay, to alleviate, or to arrest or inhibit development ofthe symptoms or conditions associated with diseases (e.g. cancers). Theterm “therapeutic effect” refers to the reduction, elimination, orprevention of the disease, symptoms of the disease, or side effects ofthe disease in the subject.

The term “effective amount” as used herein means the amount that, whenadministered to a subject for treating a disease, is sufficient toeffect treatment for that disease.

The term “subject”, as used herein, refers to any mammalian subject forwhom diagnosis, treatment or therapy is desired. “Mammal” for purposesof treatment refers to any animal classified as a mammal, includinghumans, domestic and farm animals, and laboratory, zoo, sports, or petanimals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice,rats, rabbits, guinea pigs, monkeys etc.

In an aspect, the present disclosure provides an antibody specificallybinding to ENPP3, or an antigen binding fragment thereof, comprising alight chain variable region (VL) and a heavy chain variable region (VH),wherein the VL comprises LCDRs 1-3 having the amino acid sequences asset forth in SEQ ID NOs: 1-3 respectively, and the VH comprises HCDRs1-3 having the amino acid sequences as set forth in SEQ ID NOs: 6-8respectively.

In some embodiments, CDR sequences are defined according to Kabatnumbering system.

When CDR sequences are defined according to Kabat numbering system, theVL of the antibody disclosed herein comprises LCDR1, LCDR2 and LCDR3having the amino acid sequences as set forth in SEQ ID NO: 1(SGSSSNIGNNYVS), SEQ ID NO: 2 (DNNKRPS) and SEQ ID NO: 3 (GVWDSSLRAEL)respectively, and the VH of the antibody disclosed herein comprisesHCDR1, HCDR2 and HCDR3 having the amino acid sequences as set forth inSEQ ID NO: 6 (NAWMS), SEQ ID NO: 7 (YISSSGSTIYYADSVKG) and SEQ ID NO: 8(LAGPYYFDY) respectively.

In some embodiments of the antibody or the antigen binding fragmentthereof disclosed herein, the VL comprises an amino acid sequence havingat least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100% sequence identity to SEQ ID NO: 4, and the VHcomprises an amino acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100% sequenceidentity to SEQ ID NO: 9.

In some embodiments, the VL comprises a functional variant of the aminoacid sequence as set forth in SEQ ID NO: 4 formed by insertion, deletionand/or substitution of one or more amino acid(s) therein, provided thatthe functional variant retains the ability of binding to ENPP3. In someembodiments, the VH comprises a functional variant of the amino acidsequence as set forth in SEQ ID NO: 9 formed by insertion, deletionand/or substitution of one or more amino acid(s) therein, provided thatthe functional variant retains the ability of binding to ENPP3.

The functional variant comprises or consists of an amino acid sequencehaving at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%,at least 99.7%, at least 99.8%, or at least 99.9% sequence identity tothe amino acid sequence of the parent polypeptide. For example, thefunctional variant of SEQ ID NO: 4 comprises or consists of an aminoacid sequence having at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, at least99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%,at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%sequence identity to SEQ ID NO: 4. For example, the functional variantof SEQ ID NO: 9 comprises or consists of an amino acid sequence havingat least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%,at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO:9.

In some embodiments, the functional variant of SEQ ID NO: 4 comprises orconsists of an amino acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%,or at least 99.9% sequence identity to SEQ ID NO: 4 and formed byinsertion, deletion and/or substitution of one or more amino acid(s) inSEQ ID NO: 4. In some embodiments, the functional variant of SEQ ID NO:9 comprises or consists of an amino acid sequence having at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%,at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least99.8%, or at least 99.9% sequence identity to SEQ ID NO: 9 and formed byinsertion, deletion and/or substitution of one or more amino acid(s) inSEQ ID NO: 9.

In the context of the functional variant, the number of the inserted,deleted and/or substituted amino acid is preferably no more than 40% ofthe total number of amino acids in the parent amino acid sequence, morepreferably no more than 35%, more preferably 1-33%, and more preferably5-30%, more preferably 10-25%, more preferably 15-20%. For example, thenumber of the inserted, deleted and/or substituted amino acid can be1-20, preferably 1-10, more preferably 1-7, still more preferably 1-5,and most preferably 1-2. In a preferred embodiment, the number of theinserted, deleted and/or substituted amino acid is 1, 2, 3, 4, 5, 6, or7.

In some embodiments, the insertion, deletion and/or substitution can beperformed at framework (FR) regions, e.g., at FR1, FR2, FR3, and/or FR4.

In some embodiments, the substitution of one or more amino acid(s) canbe conservative substitution of one or more amino acid(s). Suchconservative substitutions preferably are substitutions in which oneamino acid within the following groups (a)-(e) is substituted by anotheramino acid residue within the same group: (a) small aliphatic, nonpolaror slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar,negatively charged residues and their (uncharged) amides: Asp, Asn, Gluand Gln; (c) polar, positively charged residues: His, Arg and Lys; (d)large aliphatic, nonpolar residues: Met, Leu, He, Val and Cys; and (e)aromatic residues: Phe, Tyr and Trp.

Particularly preferred conservative substitutions are as follows: Alainto Gly or into Ser; Arg into Lys; Asn into Gln or into His; Asp intoGlu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro;His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or intoVal; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or intoIle; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trpinto Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into Leu.

In a preferred embodiment, the VL comprises an amino acid sequence asset forth in SEQ ID NO: 4 and the VH comprises an amino acid sequence asset forth in SEQ ID NO: 9.

Based on the amino acid sequence of heavy chain constant regions of theantibody, a immunoglobulin molecule can be divided into five classes(isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided intodifferent subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc. Thelight chain of the antibody can be classified as a lambda (λ) chain or akappa (κ) chain, based on the amino acid sequence of the light chain.The antibodies disclosed herein can be of any classes or subtypes above.

In some embodiments, the antibody can be of an isotype selected from thegroup consisting of IgG, IgA, IgM, IgE and IgD. In some embodiments, theantibody can be of a subtype selected from the group consisting of IgG1,IgG2, IgG3, and IgG4. In a preferred embodiment, the antibody is an IgG1antibody.

The antibody disclosed herein can be an intact antibody or the antigenbinding fragment thereof. The antigen binding fragment can be anyfragments of the antibody that retain the ability to specifically bindto ENPP3. Examples of antigen binding fragments include but are notlimited to a Fab fragment; a F(ab′)₂ fragment; a Fab′ fragment; a Fdfragment; a Fd′ fragment; a Fv fragment; a scFv fragment; a dAbfragment; an isolated complementarity determining region (CDR); ananobody; a linear antibody comprising a pair of tandem Fd segments(VH-CH1-VH-CH1), and a modified version of any of the foregoingfragments, which retains antigen binding activity.

In some embodiments, the antigen binding fragment can be selected fromthe group consisting of Fab, Fab′, F(ab′)₂, Fv, scFv, and ds-scFv. In apreferred embodiment, the antigen binding fragment is Fab or scFv. Inanother preferred embodiment, the antigen binding fragment is Fab.

In some embodiments, the antibody can be a monoclonal antibody. In someembodiments, the antibody comprises a light chain comprising an aminoacid sequence having at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:5 and a heavy chain comprising an amino acid sequence having at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100% sequence identity to SEQ ID NO: 10.

In some embodiments, the light chain comprises a functional variant ofthe amino acid sequence as set forth in SEQ ID NO: 5 formed byinsertion, deletion and/or substitution of one or more amino acid(s)therein, provided that the functional variant retains the ability ofbinding to ENPP3. In some embodiments, the heavy chain comprises afunctional variant of the amino acid sequence as set forth in SEQ ID NO:10 formed by insertion, deletion and/or substitution of one or moreamino acid(s) therein, provided that the functional variant retains theability of binding to ENPP3.

For example, the functional variant of SEQ ID NO: 5 comprises orconsists of an amino acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%,or at least 99.9% sequence identity to SEQ ID NO: 5. For example, thefunctional variant of SEQ ID NO: 10 comprises or consists of an aminoacid sequence having at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, at least99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%,at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%sequence identity to SEQ ID NO: 10.

In some embodiments, the number of the inserted, deleted and/orsubstituted amino acid is preferably no more than 40% of the totalnumber of amino acids in the parent amino acid sequence, more preferablyno more than 35%, more preferably 1-33%, and more preferably 5-30%, morepreferably 10⁻²⁵%, more preferably 15-20%. For example, the number ofthe inserted, deleted and/or substituted amino acid can be 1-50,preferably 1-20, more preferably 1-10, still more preferably 1-5. In apreferred embodiment, the number of the inserted, deleted and/orsubstituted amino acid is 1, 2, 3, 4, 5, 6, or 7.

In some embodiments, the insertion, deletion and/or substitution can beperformed at framework (FR) regions, e.g., at FR1, FR2, FR3 and/or FR4;and/or constant regions, e.g., CL, CH1, CH2 and/or CH3.

In some embodiments, the substitution of one or more amino acid(s) canbe conservative substitution of one or more amino acid(s). Examples ofconservative substitutions are as described above.

In a preferred embodiment, the light chain comprises an amino acidsequence as set forth in SEQ ID NO: 5 and the heavy chain comprises anamino acid sequence as set forth in SEQ ID NO: 10.

In other embodiments, the antibody can be a bispecific or amulti-specific antibody. In some embodiments, the antibody is abispecific antibody which further comprises a second antigen bindingregion binding to a second antigen. In some embodiments, the secondantigen can be a tumor associated antigen or an immune cell antigen.

Many tumor associated antigens associated with specific cancers havebeen identified in the art. In some embodiments, tumor-associatedantigens are antigens that can potentially stimulate an obvioustumor-specific immune response. Some of these antigens are encoded bynormal cells, but not necessarily expressed by normal cells. Theseantigens can be characterized as those that are usually silent (i.e.,not expressed) in normal cells, those that are expressed only duringcertain stages of differentiation, and those that are expressed overtime, such as embryonic and fetal antigens. Other cancer antigens areencoded by mutant cell genes such as oncogenes (e.g. activated rasoncogene), suppressor genes (e.g. mutant p53), and fusion proteinsproduced by internal deletions or chromosomal translocations. Othercancer antigens can be encoded by viral genes, such as those carried onRNA and DNA tumor viruses. Many other tumor associated antigens andantibodies against them are known and/or commercially available, and canalso be produced by those skilled in the art.

Examples of tumor associated antigens include but are not limited to5T4, alphafetoprotein, CA-125, carcinoembryonic antigen, CD19, CD20,CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, c-Met,CSPG4, IgM, C-type lectin-like molecule 1 (CLL-1), EGFR, EGFRvIII,epithelial tumor antigen, ERBB2, FLT3, folate binding protein, GD2, GD3,HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gp120,melanoma-associated antigen, MUC-1, mutated p53, mutated ras, ROR1,GPC3, VEGFR2, and combinations thereof.

In some embodiments, the second antigen can be a T-cell antigen. In someembodiments, the T-cell antigen can be selected from the groupconsisting of T cell receptor (TCR), CD3, CD4, CD8, CD16, CD25, CD28,CD38, CD44, CD62L, CD69, ICOS, 41-BB (CD137), and NKG2D or anycombination thereof. In some embodiments, the T-cell antigen is CD3, andthe second antigen binding region binds to any of γ chain, δ chain, εchain, ζ chain and η chain of CD3.

In some embodiments, the second antigen is CD3, and the second antigenbinding region comprises a VL and a VH, wherein the VL comprises LCDRs1-3 having the amino acid sequences as set forth in SEQ ID NOs: 11-13respectively, and the VH comprises HCDRs 1-3 having the amino acidsequences as set forth in SEQ ID NOs: 16-18 respectively.

In some embodiments, CDR sequences are defined according to Kabatnumbering system. When using Kabat defined CDR sequences, the VL of thesecond antigen binding region disclosed herein comprises LCDR1, LCDR2and LCDR3 having the amino acid sequences as shown in SEQ ID NO: 11(RSSTGAVTTSNYAN), SEQ ID NO: 12 (GANKRAP) and SEQ ID NO: 13 (ALWYSNLWV)respectively, and the VH of the second antigen binding region disclosedherein comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequencesas shown in SEQ ID NO: 16 (GFTFNTY), SEQ ID NO: 17 (RSKYNNYA) and SEQ IDNO: 18 (HGNFGSSYVSYFAY) respectively.

In some embodiments, the second antigen binding region comprises a VLcomprising an amino acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100% sequenceidentity to SEQ ID NO: 14 and a VH comprising an amino acid sequencehaving at least 80%, at least 85%, at least 90%, at least 95%, at least98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19.

In some embodiments, the VL comprises a functional variant of the aminoacid sequence as set forth in SEQ ID NO: 14 formed by insertion,deletion and/or substitution of one or more amino acid(s) therein,provided that the functional variant retains the ability of binding toCD3. In some embodiments, the VH comprises a functional variant of theamino acid sequence as set forth in SEQ ID NO: 19 formed by insertion,deletion and/or substitution of one or more amino acid(s) therein,provided that the functional variant retains the ability of binding toCD3.

For example, the functional variant of SEQ ID NO: 14 comprises orconsists of an amino acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%,or at least 99.9% sequence identity to SEQ ID NO: 14. For example, thefunctional variant of SEQ ID NO: 19 comprises or consists of an aminoacid sequence having at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, at least99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%,at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%sequence identity to SEQ ID NO: 19.

In some embodiments, the number of the inserted, deleted and/orsubstituted amino acid is preferably no more than 40% of the totalnumber of amino acids in the parent amino acid sequence, more preferablyno more than 35%, more preferably 1-33%, and more preferably 5-30%, morepreferably 10⁻²⁵%, more preferably 15-20%. For example, the number ofthe inserted, deleted and/or substituted amino acid can be 1-20,preferably 1-10, more preferably 1-7, still more preferably 1-5, andmost preferably 1-2. In a preferred embodiment, the number of theinserted, deleted and/or substituted amino acid is 1, 2, 3, 4, 5, 6, or7.

In some embodiments, the insertion, deletion and/or substitution can beperformed at framework (FR) regions, e.g., at FR1, FR2, FR3, and/or FR4.

In some embodiments, the substitution of one or more amino acid(s) canbe conservative substitution of one or more amino acid(s). Examples ofconservative substitutions are as described above.

In a preferred embodiment, the second antigen binding region comprises aVL comprising an amino acid sequence as set forth in SEQ ID NO: 14 and aVH comprising an amino acid sequence as set forth in SEQ ID NO: 19.

In some embodiments, the VL of the second antigen binding region islinked to the C-terminal of the VL of the antibody specifically bindingto ENPP3, optionally via a first linker, and the VH of the secondantigen binding region is linked to the C-terminal of the VH of theantibody specifically binding to ENPP3, optionally via a second linker,wherein the first linker and the second linker are the same ordifferent.

In some embodiments, each of the first linker and the second linkerindependently comprises an amino acid selected from SEQ ID NO: 21(GSGGGGSGGGGS) and SEQ ID NO: 22 (GSGGSGGGGSGGGGS). In some embodiments,the first linker comprises an amino acid sequence as set forth in SEQ IDNO: 21 (GSGGGGSGGGGS), and the second linker comprises an amino acidsequence as set forth in SEQ ID NO: 22 (GSGGSGGGGSGGGGS).

In some embodiments, the bispecific antibody comprises a light chaincomprising an amino acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100% sequenceidentity to SEQ ID NO: 15 and a heavy chain comprising an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 20.

In some embodiments, the light chain comprises a functional variant ofthe amino acid sequence as set forth in SEQ ID NO: 15 formed byinsertion, deletion and/or substitution of one or more amino acid(s)therein, provided that the functional variant retains the ability ofbinding to ENPP3 and CD3. In some embodiments, the heavy chain comprisesa functional variant of the amino acid sequence as set forth in SEQ IDNO: 20 formed by insertion, deletion and/or substitution of one or moreamino acid(s) therein, provided that the functional variant retains theability of binding to ENPP3 and CD3.

For example, the functional variant of SEQ ID NO: 15 comprises orconsists of an amino acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%,or at least 99.9% sequence identity to SEQ ID NO: 15. For example, thefunctional variant of SEQ ID NO: 20 comprises or consists of an aminoacid sequence having at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, at least99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%,at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%sequence identity to SEQ ID NO: 20.

In some embodiments, the number of the inserted, deleted and/orsubstituted amino acid is preferably no more than 40% of the totalnumber of amino acids in the parent amino acid sequence, more preferablyno more than 35%, more preferably 1-33%, and more preferably 5-30%, morepreferably 10⁻²⁵%, more preferably 15-20%. For example, the number ofthe inserted, deleted and/or substituted amino acid can be 1-50,preferably 1-20, more preferably 1-10, still more preferably 1-5. In apreferred embodiment, the number of the inserted, deleted and/orsubstituted amino acid is 1, 2, 3, 4, 5, 6, or 7.

In some embodiments, the insertion, deletion and/or substitution can beperformed at framework (FR) regions, e.g., at FR1, FR2, FR3 and/or FR4;and/or constant regions, e.g., CL, CH1, CH2 and/or CH3.

In some embodiments, the substitution of one or more amino acid(s) canbe conservative substitution of one or more amino acid(s). Examples ofconservative substitutions are as described above.

In a preferred embodiment, the light chain comprises an amino acidsequence as set forth in SEQ ID NO: 15 and the heavy chain comprises anamino acid sequence as set forth in SEQ ID NO: 20.

In some embodiments, the bispecific antibody can be a bispecific T-cellengager (BITE®). In some embodiments of the antibody or the antigenbinding fragment thereof disclosed herein, the bispecific antibody is inform of an HBiTE as described in PCT application No. PCT/US2018/016524(which is incorporated herein by reference in its entirety). In theHBiTE, the light chain, from N-terminus to C-terminus, comprises ananti-target VL domain, an anti-CD3 VL-CL and a monomeric human IgG1 Fc(e.g., mFc7.2); and the heavy chain, from N-terminus to C-terminus,comprises an anti-target VH domain, an anti-CD3 VH-CH1 and a monomerichuman IgG1 Fc (e.g., mFc7.2). Monomeric Fc7.2 contains two amino acidmutations (T366L and Y407H) capable of inhibiting Fc homodimerization.

In another aspect, the present disclosure provides a bispecific antibodyor an antigen binding fragment thereof, comprising a first antigenbinding region binding to ENPP3 comprising a VL and a VH and a secondantigen binding region binding to CD3 comprising a VL and a VH, whereinthe VL of the first antigen binding region comprises LCDRs 1-3 havingthe amino acid sequences as set forth in SEQ ID NOs: 1-3 respectively,and the VH of the first antigen binding region comprises HCDRs 1-3having the amino acid sequences as set forth in SEQ ID NOs: 6-8respectively; and the VL of the second antigen binding region comprisesLCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs:11-13 respectively, and the VH of the second antigen binding regioncomprises HCDRs 1-3 having the amino acid sequences as set forth in SEQID NOs: 16-18 respectively.

In some embodiments of the bispecific antibody or the antigen bindingfragment thereof disclosed herein, the VL of the first antigen bindingregion comprises an amino acid sequence having at least 80%, at least85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 4 and the VH of the first antigenbinding region comprises an amino acid sequence having at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100% sequence identity to SEQ ID NO: 9; and the VL of the second antigenbinding region comprises an amino acid sequence having at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100% sequence identity to SEQ ID NO: 14 and the VH of the second antigenbinding region comprises an amino acid sequence having at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100% sequence identity to SEQ ID NO: 19.

In some embodiments, the VL of the first antigen binding regioncomprises a functional variant of the amino acid sequence as set forthin SEQ ID NO: 4 formed by insertion, deletion and/or substitution of oneor more amino acid(s) therein, provided that the functional variantretains the ability of binding to ENPP3. In some embodiments, the VH ofthe first antigen binding region comprises a functional variant of theamino acid sequence as set forth in SEQ ID NO: 9 formed by insertion,deletion and/or substitution of one or more amino acid(s) therein,provided that the functional variant retains the ability of binding toENPP3. In some embodiments, the VL of the second antigen binding regioncomprises a functional variant of the amino acid sequence as set forthin SEQ ID NO: 14 formed by insertion, deletion and/or substitution ofone or more amino acid(s) therein, provided that the functional variantretains the ability of binding to CD3. In some embodiments, the VH ofthe second antigen binding region comprises a functional variant of theamino acid sequence as set forth in SEQ ID NO: 19 formed by insertion,deletion and/or substitution of one or more amino acid(s) therein,provided that the functional variant retains the ability of binding toCD3.

The functional variants of SEQ ID NOs: 4, 9, 14 and 19 can be those asdescribed above.

In a preferred embodiment, the VL of the first antigen binding regioncomprises an amino acid sequence as set forth in SEQ ID NO: 4 and the VHof the first antigen binding region comprises an amino acid sequence asset forth in SEQ ID NO: 9; and the VL of the second antigen bindingregion comprises an amino acid sequence as set forth in SEQ ID NO: 14and the VH of the second antigen binding region comprises an amino acidsequence as set forth in SEQ ID NO: 19.

In some embodiments, the VL of the second antigen binding region islinked to the C-terminal of the VL of the first antigen binding region,optionally via a first linker, and the VH of the second antigen bindingregion is linked to the C-terminal of the VH of the first antigenbinding region, optionally via a second linker, wherein the first linkerand the second linker are the same or different. In some embodiments,each of the first linker and the second linker independently comprisesan amino acid selected from SEQ ID NO: 21 (GSGGGGSGGGGS) and SEQ ID NO:22 (GSGGSGGGGSGGGGS). In some embodiments, the first linker comprises anamino acid sequence as set forth in SEQ ID NO: 21 (GSGGGGSGGGGS), andthe second linker comprises an amino acid sequence as set forth in SEQID NO: 22 (GSGGSGGGGSGGGGS).

In some embodiments, the bispecific antibody comprises a singlepolypeptide chain comprising the first antigen binding region and thesecond antigen binding region, and optionally an Fc region.

The Fc region may be of any isotype, including, but not limited to,IgG1, IgG2, IgG3 and IgG4, and may comprise one or more mutations ormodifications. In one embodiment, the Fc region is of IgG1 isotype orderived therefrom, optionally with one or more mutations ormodifications. In one embodiment, the Fc region is human IgG1 Fc.

In one embodiment, the Fc region is effector-function-deficient. Forexample, the Fc region may be of an IgG1 isotype, or a non-IgG1 type,e.g. IgG2, IgG3 or IgG4, which has been mutated such that the ability tomediate effector functions, such as ADCC, has been reduced or eveneliminated. Such mutations have e.g. been described in Dall'Acqua W F etal., J Immunol. 177(2):1129-1138 (2006) and Hezareh M, J Virol.;75(24):12161-12168 (2001).

In one embodiment, the Fc region comprises a mutation removing theacceptor site for Asn-linked glycosylation or is otherwise manipulatedto change the glycosylation properties. For example, in an IgG1 Fcregion, an N297Q mutation can be used to remove an Asn-linkedglycosylation site. Accordingly, in a specific embodiment, Fc regioncomprises an IgG1 sequence with an N297Q mutation.

In a further embodiment, the Fc region is glyco-engineered to reducefucose and thus enhance ADCC, e.g. by addition of compounds to theculture media during antibody production as described in US2009317869 oras described in van Berkel et al. (2010) Biotechnol. Bioeng. 105:350 orby using FUT8 knockout cells, e.g. as described in Yamane-Ohnuki et al.(2004) Biotechnol. Bioeng 87:614. ADCC may alternatively be optimizedusing the method described by Umaña et al. (1999) Nature Biotech 17:176.In a further embodiment, the Fc region has been engineered to enhancecomplement activation, e.g. as described in Natsume et al. (2009) CancerSci. 100:2411.

In some embodiments, the Fc region comprises modifications or mutationsthat can inhibit Fc homodimerization. In some embodiments, the Fc regioncomprises a variant of a human IgG1 Fc wildtype sequence. The variantcan comprise amino acid substitutions at positions T366 and Y407 ofhuman IgG1 (Kabat numbering). Preferably, T366 is substituted with L(Leucine). Preferably, Y407 is substituted with I(Isoleucine),F(Phenylalanine), L(Leucine), M(Methionine), H(Histidine), K(Lysine),S(Serine), Q(Glutamine), T(Threonine), W(Tryptophan), A(Alanine),G(Glycine) or N (Asparagine). More preferably, Y407 is substituted withH. In one embodiment, T366 is substituted with L, and Y407 issubstituted with H.

In some embodiments, the Fc region can be a monomeric human IgG1 Fc(e.g., mFc7.2) as described in PCT application No. PCT/US2018/016524,which is incorporated herein by reference in its entirety.

In some embodiments, the bispecific antibody comprises a firstpolypeptide chain comprising the VL of the first antigen binding regionand the VL of the second antigen binding region, and optionally an Fcregion; and a second polypeptide chain comprising the VH of the firstantigen binding region and the VH of the second antigen binding region,and optionally an Fc region. The Fc region can be those as describeabove.

In some embodiments, the first polypeptide chain further comprises alight chain constant region (CL). In some embodiments, the firstpolypeptide chain comprises a monomeric human IgG1 Fc (e.g., mFc7.2) asdescribed above. In some embodiments, the first polypeptide chaincomprises, from N-terminal to C-terminal: the VL of the first antigenbinding region, the VL of the second antigen binding region, CL andmFc7.2.

In some embodiments, the second polypeptide chain further comprises aheavy chain constant region (CH), e.g., CH1. In some embodiments, thefirst polypeptide chain comprises a monomeric human IgG1 Fc (e.g.,mFc7.2) as described above. In some embodiments, the second polypeptidechain comprises, from N-terminal to C-terminal: the VH of the firstantigen binding region, the VH of the second antigen binding region, CH1and mFc7.2.

In some embodiments, the bispecific antibody comprises a light chaincomprising an amino acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100% sequenceidentity to SEQ ID NO: 15 and a heavy chain comprising an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 20.

In some embodiments, the light chain comprises a functional variant ofthe amino acid sequence as set forth in SEQ ID NO: 15 formed byinsertion, deletion and/or substitution of one or more amino acid(s)therein, provided that the functional variant retains the ability ofbinding to ENPP3 and CD3. In some embodiments, the heavy chain comprisesa functional variant of the amino acid sequence as set forth in SEQ IDNO: 20 formed by insertion, deletion and/or substitution of one or moreamino acid(s) therein, provided that the functional variant retains theability of binding to ENPP3 and CD3.

The functional variants of SEQ ID NOs: 15 and 20 can be those asdescribed above.

In a preferred embodiment, the light chain comprises an amino acidsequence as set forth in SEQ ID NO: 15 and the heavy chain comprises anamino acid sequence as set forth in SEQ ID NO: 20.

In some embodiments, the bispecific antibody can be a bispecific T-cellengager (BITE®), preferably an HBiTE as described above.

In still another aspect, the present disclosure provides a nucleic acidcomprising a nucleotide sequence encoding the antibody or the antigenbinding fragment thereof disclosed herein or the bispecific antibody orthe antigen binding fragment thereof disclosed herein.

In yet another aspect, the present disclosure provides a vectorcomprising the nucleic acid disclosed herein.

Any vector may be suitable for the present disclosure. In someembodiments, the vector is a viral vector. In some embodiments, thevector is a retroviral vector, a DNA vector, a murine leukemia virusvector, an SFG vector, a plasmid, a RNA vector, an adenoviral vector, abaculoviral vector, an Epstein Barr viral vector, a papovaviral vector,a vaccinia viral vector, a herpes simplex viral vector, an adenovirusassociated vector (AAV), a lentiviral vector, or any combinationthereof. Suitable exemplary vectors include e.g., pBY, pGAR, pBABE-puro,pBABE-neo largeTcDNA, pBABE-hygro-hTERT, pMKO.1 GFP, MSCV-IRES-GFP,pMSCV PIG (Puro IRES GFP empty plasmid),pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES Luciferase, pMIG,MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre,pRXTN, pLncEXP, and pLXIN-Luc.

A recombinant expression vector may be any suitable recombinantexpression vector. Suitable vectors comprise those designed forpropagation and expansion or for expression or both, such as plasmidsand viruses. For example, a vector may be selected from the pUC series(Fermentas Life Sciences, Glen Burnie, Md.), the pBluescript series(Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.),the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series(Clontech, Palo Alto, Calif.). Bacteriophage vectors, such as λGT10,λGT11, λZapII (Stratagene), λEMBL4, and λNM1149, also may be used.Examples of plant expression vectors useful in the context of thedisclosure comprise pBY, pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19(Clontech). Examples of animal expression vectors useful in the contextof the disclosure comprise pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).

Recombinant expression vectors may be prepared using standardrecombinant DNA techniques described in, for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring HarborPress, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates and JohnWiley & Sons, N Y, 1994. Constructs of expression vectors, which arecircular or linear, may be prepared to contain a replication systemfunctional in a prokaryotic or eukaryotic host cell. Replication systemsmay be derived, e.g., from ColEl, 2μ plasmid, λ, SV40, bovine papillomavirus, and the like.

In another aspect, the present disclosure provides a host cellcomprising the nucleic acid disclosed herein or the vector disclosedherein.

Any cell may be used as a host cell for the nucleic acids or the vectorsof the present disclosure. In some embodiments, the cell can be aprokaryotic cell, fungal cell, yeast cell, or higher eukaryotic cellssuch as a mammalian cell. Suitable prokaryotic cells include, withoutlimitation, eubacteria, such as Gram-negative or Gram-positiveorganisms, for example, Enterobactehaceae such as Escherichia, e.g., E.coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, e.g.,Salmonella typhimurium, Serratia, e.g., Serratia marcescans, andShigella; Bacilli such as B. subtilis and B. licheniformis; Pseudomonassuch as P. aeruginosa; and Streptomyces. In some embodiments, the cellis a human cell. In some embodiments, the cell is an immune cell. Insome embodiments, host cells include, for example, CHO cells, such asCHOS cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293Tand HEK293FS.

In still another aspect, the present disclosure provides apharmaceutical composition comprising (i) the antibody or the antigenbinding fragment thereof disclosed herein, or the bispecific antibody orthe antigen binding fragment thereof disclosed herein; and (ii) apharmaceutically acceptable carrier or excipient.

In some embodiments, the carrier or excipient for use with thecomposition disclosed herein includes but is not limited to maleic acid,tartaric acid, lactic acid, citric acid, acetic acid, sodiumbicarbonate, sodium phosphate, histidine, glycine, sodium chloride,potassium chloride, calcium chloride, zinc chloride, water, dextrose,N-methylpyrrolidone, dimethyl sulfoxide, N,N-dimethylacetamide, ethanol,propylene glycol, polyethylene glycol, diethylene glycol monoethylether, and surfactant polyoxyethylene-sorbitan monooleate.

In some embodiments of the pharmaceutical composition disclosed herein,the pharmaceutical composition further comprises a second therapeuticagent. In some embodiments, the second therapeutic agent can be selectedfrom an antibody, a chemotherapeutic agent and a small molecule drug. Insome embodiments, the second therapeutic agent can be selected from aBruton's tyrosine kinase (BTK) inhibitor, a PI3K inhibitor, a HDACinhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1/PD-L1 inhibitor, aLAGS inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor,and glucocorticoid, or any combination thereof.

In some embodiments, the therapeutic agent is a chemotherapeutic agent.The chemotherapeutic agents can include, for example, cytotoxic agents,anti-metabolite agents (e.g., folate antagonists, purine analogs,pyrimidine analogs, etc.), topoisomerase inhibitors (e.g., camptothecinderivatives, anthracenedione, anthracyclines, epipodophyllotoxins,quinoline alkaloids, etc.), anti-microtubule agents (e.g., taxanes,vinca alkaloids), protein synthesis inhibitors (e.g., cephalotaxine,camptothecin derivatives, quinoline alkaloids), alkylating agents (e.g.,alkyl sulfonates, ethylenimines, nitrogen mustards, nitrosoureas,platinum derivatives, triazenes, etc.), alkaloids, terpenoids, andkinase inhibitors.

In yet another aspect, the present disclosure provides a conjugatecomprising the antibody or the antigen binding fragment thereofdisclosed herein or the bispecific antibody or the antigen bindingfragment thereof disclosed herein, and a chemical moiety conjugatedthereto.

In some embodiments of the conjugate disclosed herein, the chemicalmoiety is selected from the group consisting of a therapeutic agent, adetectable moiety, and an immune stimulatory molecule.

In some embodiments, the therapeutic agent includes but is not limitedto immunomodulators, radioactive compounds, enzymes (for exampleperforin), chemotherapeutic agents (for example cis-platin), or a toxin.In some embodiments, the therapeutic agent can be such as maytansine,geldanamycin, tubulin inhibitors such as tubulin binding agents (e.g.,auristatins), or minor groove binding agents such as calicheamicin.

Other suitable therapeutic agents include such as, small moleculecytotoxic agents, i.e. compounds with the ability to kill mammaliancells having a molecular weight of less than 700 Daltons. Such compoundscould also contain toxic metals capable of having a cytotoxic effect.Furthermore, it is to be understood that these small molecule cytotoxicagents also include pro-drugs, i.e. compounds that decay or areconverted under physiological conditions to release cytotoxic agents.Examples of such agents include cis-platin, maytansine derivatives,rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine,ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimersodiumphotofrin II, temozolomide, topotecan, trimetreate glucuronate,auristatin E vincristine and doxorubicin; peptide cytotoxins, i.e.proteins or fragments thereof with the ability to kill mammalian cells,for example, ricin, diphtheria toxin, pseudomonas bacterial exotoxin A,DNase and RNase; radio-nuclides, i.e. unstable isotopes of elementswhich decay with the concurrent emission of one or more of a or βparticles, or γ rays, for example, iodine-131, rhenium-186, indium-111,yttrium-90, bismuth-210, bismuth-213, actinium-225 and astatine-213;chelating agents may be used to facilitate the association of theseradionuclides to the molecules, or multimers thereof.

In some embodiments, the detectable moiety can be selected from thegroup consisting of biotin, streptavidin, an enzyme or catalyticallyactive fragment thereof, a radionuclide, a nanoparticle, a paramagneticmetal ion, or a fluorescent, phosphorescent, or chemiluminescentmolecule. A detectable moiety for diagnostic purposes include forinstance, fluorescent labels, radiolabels, enzymes, nucleic acid probesand contrast reagents.

In some embodiments, the immune stimulatory molecule is an immuneeffector molecules which stimulate immune response. For example, theimmune stimulatory molecule can be cytokines such as IL-2 and IFN-γ,chemokines such as IL-8, platelet factor 4, melanoma growth stimulatoryprotein, complement activators; viral/bacterial protein domains, orviral/bacterial peptides.

In another aspect, the present disclosure provides a method of treatinga cancer in a subject comprising administering to the subject aneffective amount of the antibody or the antigen binding fragment thereofdisclosed herein, the bispecific antibody or the antigen bindingfragment thereof disclosed herein, the pharmaceutical compositiondisclosed herein, or the conjugate disclosed herein.

In some embodiments of the method disclosed herein, the cancer is anENPP3 positive cancer. In some embodiments, the cancer can be selectedfrom the group consisting of mastocytosis, leukemia, liver cancer, coloncancer, rectal cancer, colorectal cancer, bile duct cancer, Wilms tumor,pancreatic cancer, breast cancer, lung cancer, ovarian cancer,esophageal cancer, bladder cancer, prostate cancer, colorectal cancer,uterine cancer, cervical cancer, brain cancer, cervical cancer, gastriccancer, cholangiocarcinoma, chondrosarcoma, kidney cancer, thyroidcancer, skin cancer, glioma, neuroblastoma, lymphoma and myeloma.Preferably, the cancer is selected from the group consisting ofmastocytosis, leukemia, kidney cancer (e.g. renal cell carcinoma), lungcancer, gastric cancer, ovarian cancer, breast cancer, pancreaticcancer, colon cancer, rectal cancer, colorectal cancer, bile ductcancer, liver cancer (e.g. hepatocellular carcinoma), and Wilms tumor.

In some embodiments, dosage administered to a subject may vary with theembodiment, the medicament employed, the method of administration, andthe site and subject being treated. However, a dose should be sufficientto provide a therapeutic response. A clinician may determine theeffective amount to be administered to a human or other subject in orderto treat a medical condition. The precise amount required to betherapeutically effective may depend upon numerous factors, e.g., suchas the activity of the antibody, and the route of administration.

A dose of the antibodies, compositions or conjugates described hereinmay be administered to a mammal at one time or in a series of sub-dosesadministered over a suitable period of time, e.g., on a daily,semi-weekly, weekly, bi-weekly, semi-monthly, bi-monthly, semi-annual,or annual basis, as needed. A dosage unit comprising an effective amountof antibodies, compositions or conjugates may be administered in asingle daily dose, or the total daily dosage may be administered in two,three, four, or more divided doses administered daily, as needed.

A suitable means of administration may be selected by a medicalpractitioner. Route of administration may be parenteral, for example,administration by injection, transnasal administration, transpulmonaryadministration, or transcutaneous administration. Administration may besystemic or local by intravenous injection, intramuscular injection,intraperitoneal injection, subcutaneous injection. In some embodiments,the antibodies, compositions or conjugates are selected for parenteraldelivery, for inhalation, or for delivery through the digestive tract,such as orally. Dose and method of administration may vary depending onthe weight, age, condition, and the like of the subject, and may besuitably selected.

In some embodiments, the method further comprises administering to thesubject a second therapeutic agent. In certain embodiments, theantibody, composition or conjugate disclosed herein is administeredprior to, substantially simultaneously with, or after the administrationof the second therapeutic agent.

In some embodiments, the second therapeutic agent is selected from anantibody, a chemotherapeutic agent and a small molecule drug. In somepreferred embodiments, the second therapeutic agent can be selected froma Bruton's tyrosine kinase (BTK) inhibitor, a PI3K inhibitor, a HDACinhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1/PD-L1 inhibitor, aLAGS inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor,and glucocorticoid, or any combination thereof.

In some embodiments, the second therapeutic agent is a chemotherapeuticagent. The chemotherapeutic agents can include, for example, cytotoxicagents, anti-metabolite agents (e.g., folate antagonists, purineanalogs, pyrimidine analogs, etc.), topoisomerase inhibitors (e.g.,camptothecin derivatives, anthracenedione, anthracyclines,epipodophyllotoxins, quinoline alkaloids, etc.), anti-microtubule agents(e.g., taxanes, vinca alkaloids), protein synthesis inhibitors (e.g.,cephalotaxine, camptothecin derivatives, quinoline alkaloids),alkylating agents (e.g., alkyl sulfonates, ethylenimines, nitrogenmustards, nitrosoureas, platinum derivatives, triazenes, etc.),alkaloids, terpenoids, and kinase inhibitors.

In still another aspect, the present disclosure provides a method ofdetecting ENPP3 positive cancer in a subject comprising (i) contacting asample obtained from the subject with the antibody or the antigenbinding fragment thereof disclosed herein, the bispecific antibody orthe antigen binding fragment thereof disclosed herein, or the conjugatedisclosed herein; and (ii) detecting binding of the antibody or theantigen binding fragment thereof to ENPP3 in the sample.

In some embodiments, the antibody or the antigen binding fragmentthereof is linked to a detectable moiety. The detectable moiety can beselected from the group consisting of biotin, streptavidin, an enzyme orcatalytically active fragment thereof, a radionuclide, a nanoparticle, aparamagnetic metal ion, or a fluorescent, phosphorescent, orchemiluminescent molecule. A detectable moiety for diagnostic purposesinclude for instance, fluorescent labels, radiolabels, enzymes, nucleicacid probes and contrast reagents.

In some embodiments, the cancer is an ENPP3 positive cancer. Preferably,the cancer is selected from the group consisting of mastocytosis,leukemia, kidney cancer (e.g. renal cell carcinoma), lung cancer,gastric cancer, ovarian cancer, breast cancer, pancreatic cancer, coloncancer, rectal cancer, colorectal cancer, bile duct cancer, liver cancer(e.g. hepatocellular carcinoma), and Wilms tumor.

In yet another aspect, the present disclosure provides a pharmaceuticalpack or kit comprising one or more containers filled with one or more ofthe ingredients of the pharmaceutical compositions described herein,such as the antibodies or the antigen binding fragment disclosed herein.Optionally, associated with such container(s) can be a notice in theform prescribed by a governmental agency regulating the manufacture, useor sale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

In a specific embodiment, the kit comprises a first container containingthe antibodies or the antigen binding fragment disclosed herein. In aspecific embodiment, the kit comprises a first container that is a vialcontaining the antibodies or the antigen binding fragment as alyophilized sterile powder under vacuum, and the kit further comprises asecond container comprising a pharmaceutically acceptable fluid.

In a specific embodiment, provided herein is an injection devicecontaining the antibodies or the antigen binding fragment disclosedherein. In a specific embodiment, the injection device comprises theantibody in sterile solution. In a specific embodiment, the injectiondevice is a syringe.

In still another aspect, the present disclosure provides a kit fordetecting the presence of an ENPP3 antigen in a sample comprising theantibody or the antigen binding fragment thereof disclosed herein, thebispecific antibody or the antigen binding fragment thereof disclosedherein, or the conjugate disclosed herein. Preferably, the antibody orthe antigen binding fragment thereof is linked to a detectable moiety.The detectable moiety can be selected from the group consisting ofbiotin, streptavidin, an enzyme or catalytically active fragmentthereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, or afluorescent, phosphorescent, or chemiluminescent molecule. A detectablemoiety for diagnostic purposes include for instance, fluorescent labels,radiolabels, enzymes, nucleic acid probes and contrast reagents.

In another aspect, the present disclosure provides use of the antibodyor the antigen binding fragment thereof disclosed herein, the bispecificantibody or the antigen binding fragment thereof disclosed herein, thepharmaceutical composition disclosed herein, or the conjugate disclosedherein in the manufacture of a medicament for treating a cancer in asubject. In some embodiments, the cancer is an ENPP3 positive cancer.

In still another aspect, the present disclosure provides the antibody orthe antigen binding fragment thereof disclosed herein, the bispecificantibody or the antigen binding fragment thereof disclosed herein, thepharmaceutical composition disclosed herein, or the conjugate disclosedherein for use in treating a cancer in a subject. In some embodiments,the cancer is an ENPP3 positive cancer.

In yet another aspect, the present disclosure provides use of theantibody or the antigen binding fragment thereof disclosed herein, thebispecific antibody or the antigen binding fragment thereof disclosedherein, or the conjugate disclosed herein in the manufacture of a kitfor detecting ENPP3 positive cancer in a subject.

In still another aspect, the present disclosure provides the antibody orthe antigen binding fragment thereof disclosed herein, the bispecificantibody or the antigen binding fragment thereof disclosed herein, orthe conjugate disclosed herein for use in detecting ENPP3 positivecancer in a subject.

In some embodiments of the use disclosed herein, the ENPP3 positivecancer is preferably selected from the group consisting of mastocytosis,leukemia, kidney cancer (e.g. renal cell carcinoma), lung cancer,gastric cancer, ovarian cancer, breast cancer, pancreatic cancer, coloncancer, rectal cancer, colorectal cancer, bile duct cancer, liver cancer(e.g. hepatocellular carcinoma), and Wilms tumor.

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. The present examples, along with the methodsdescribed herein are presently representative of preferred embodiments,are exemplary, and are not intended as limitations on the scope of theinvention. Changes therein and other uses which are encompassed withinthe spirit of the invention as defined by the scope of the claims willoccur to those skilled in the art.

Cell lines including HepG2 (human liver cancer cell line), LS174T (humancolorectal cancer cell line) Jrukat (human lymphoma cell line) andSK-Nep-1 (human Wilms tumor cell line) were purchased from NationalCollection of Authenticated Cell Cultures.

A tumor cell line stably expressing ENPP3, LS174T-ENPP3, was generatedby transient transfection of the commercial ENPP3 recombinant plasmidpCMV-human ENPP3 (Sino Biological) into LS174T cells using the agentLipofectamine™ LTX Reagent with PLUS™ Reagent (Thermo) andtransfection-specific media Opti-MEM™ I (Gibco). Cell culture wassupplemented with hygromycin B and positive clones were selected. After3-5 weeks, single positive clones were gradually separated and verifiedwith flow-cytometry.

Human ENPP3 (CD203c) protein was purchased from ACROBiosystems.Anti-human IgG (γ-chain specific)-R-PE antibody, anti-human IgG(Fc-specific)-peroxidase antibody and monoclonal anti-Flag®M2-peroxidasewere purchased from Sigma. M13KO7 helper phage was purchased fromInvitrogen. Dynabeads™ Myone™ Streptavidin T1 was purchased fromThermoFisher Scientific. PE anti-His tag antibody was purchased fromBioLegend.

Example 1. Panning and Screening of a Phage-Display Naive Human FabLibrary for Identification of ENPP3 Antibodies

Two large (size, 1011) phage-display naive human Fab libraries withperipheral blood B cells from about 30 healthy individuals were used forselection of antibodies against recombinant human ENPP3 conjugated tomagnetic beads (Dynabeads™ Myone™ Streptavidin T1; ThermoFisherScientific) as described previously (Zhu et al., J Virol 2006,80:891-899) with minor modification that 5, 1, 0.2 and 0.2 mg of antigenwas used in the first, second, third and fourth round of panning,respectively. After 4 rounds of biopanning, strong positive signals wereobserved by using polyclonal phage ELISA. The 4th round phage wassubsequently subjected to test for specific binding to ENPP3. By solubleexpression-based monoclonal enzyme-linked immunosorbent assay (SemELISA)and DNA sequencing analysis, a specific Fab clone, designated as 3A10,was identified. The Fab clone has a λ light chain.

The hexahistidine (HHHHHH, SEQ ID NO: 35)-tagged 3A10 Fab was expressedin E. coli strain HB2151 and purified from the soluble fraction ofperiplasm by using the Ni-NTA resin. Then ELISA was performed by usingstandard protocols to measure binding affinity to recombinant humanENPP3 (full-length extracellular domain). Briefly, the recombinant humanENPP3 (ACROBiosystems) was coated on Corning EIA/RIA high-binding96-well plates (Corning Inc.) at 200 ng per well overnight at 4° C. andblocked with 3% nonfat milk in PBS (pH7.4). Five-fold serially dilutedantibodies were added accordingly and then incubated at room temperaturefor 2 h. The plates were washed with PBS containing 0.05% TWEEN® 20.Bound antibodies were detected by HRP-conjugated anti-FLAG tag antibody(Sino Biological). The assay was developed at room temperature with TMBsubstrate (Solarbio) and OD value was measured at 450 nm with amicroplate reader.

The result showed that Fab 3A10 has a binding affinity with EC₅₀ of 9.17nM to human ENPP3 (FIG. 1 ), which is appropriate for therapeuticmonoclonal antibody development and the construction of BiTE® typeantibodies as well. In following examples, the Fab 3A10 sequence wasused for the construction of monoclonal and bispecific antibodies.

Example 2. Construction and Characterization of Anti-ENPP3 MonoclonalAntibody

Fab clone 3A10 was used to construct an intact form of monoclonalantibody IgG1 against human ENPP3 (3A10 mAb). The heavy chain Fdfragment of 3A10 Fab was fused to the N-terminus of human IgG1 Fcfragment. Each of the light chain and heavy chain was constructed intothe vector pBY separately. Construction and initial characterization ofthe 3A10 mAb were performed as follows.

Cloning of Anti-ENPP3 Monoclonal Antibody

To generate the construct of anti-ENPP3 monoclonal antibody, followingprimers were used:

-   -   pBY-SP-FP-Not1:

(SEQ ID NO: 23) 5′GAATGCGGCCGCAAACTACAAGACAGACTTGCAAAAGAAGGCATGCACAGCTCAGCACTGCTCTGTTG 3′ (sense); BI-ENPP3-3A10-VH-FP: (SEQ ID NO: 24)5′TCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCGAGGTGCAGCTGGTGGA 3′ (sense); ENPP3-3A10-Mab-VH-RP-OL: (SEQ ID NO: 25)5′CGGTGGGCATGTGTGAGTTTTGTCACAAGATTTGGGCTCAACTTTCT 3′  (antisense);BI- ENPP3-3A10-VL-FP: (SEQ ID NO: 26)5′TCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCCAGTCTGTCGTGACGCAGC 3′ (sense); ENPP3-3A10-Mab-VL-RP-Xba1: (SEQ ID NO: 27)5′CGATTCTAGAATCATGAACATTCTGTAGGGGCCACTGTCTTC 3′ (antisense); FC-FP-OL:(SEQ ID NO: 28) 5′GACAAAACTCACACATGCCCACCG 3′ (sense); Fc-RP-Xba1:(SEQ ID NO: 29) 5′CGATTCTAGAATCATTTACCCGGGGACAGGGAGAGGCT 3′ (antisense).

For the generation of light chain, the gene fragment of light chain wasamplified from anti-ENPP3 Fab 3A10 with the primer pairBI-ENPP3-3A10-VL-FP/ENPP3-3A10-Mab-VL-RP-Xba1. A gene fragment encodinga leader peptide, which helps the expression of the target gene, wasfused to the 5′ end of the light chain PCR fragment by overlapping PCRby using the primer pair pBY-SP-FP-Not1/ENPP3-3A10-Mab-VL-RP-Xba1. ThePCR product was cloned into a pBY vector by using restriction enzymesNot1/Xba1.

For the generation of heavy chain, the VH-CH1 gene fragment wasamplified from anti-ENPP3 Fab 3A10 with the primer pairBI-ENPP3-3A10-VH-FP/ENPP3-3A10-Mab-VH-RP-OL. The PCR product was fusedto the 3′ end of a gene fragment of a signal peptide by overlapping PCRusing the primer pair pBY-SP-FP-Not1/ENPP3-3A10-Mab-VH-RP-OL. The Fcdomain was amplified from an irrelevant human IgG1 containing wild typeFc gene sequence with the primer pair FC-FP-OL/Fc-RP-Xba1. To obtain thefull-length heavy chain, these two gene fragments were fused together byoverlapping PCR using the primer pair pBY-SP-FP-Not1/Fc-RP-Xba1. Thefull-length heavy chain gene fragment was then cloned into the pBYplasmid via Not1 and Xba1 restriction sites.

Protein Expression, Purification and Initial Characterization

Anti-ENPP3 3A10 mAb was expressed in 293FS cells. The plasmids andtransfection agent PEI were mixed at the ratio of 1:3 and then dropwiseadded into 293FS cell culture. The cells were continued to grow for 5-7days after transfection. The cell culture was harvested bycentrifugation at 8000 rpm for 20 min. The culture supernatantcontaining target proteins was loaded onto Gravity EshmunoA Column(Merck), and purified according to the manufacturer's instructions.

The purified proteins were subjected to SDS-PAGE. On a non-reducingSDS-PAGE, 3A10 mAb displays an apparent molecular weight (aMW) ofapproximately 150 kDa. On a reducing SDS-PAGE, the heavy chain and lightchain have an apparent molecular weight of approximately 50 kDa and 25kDa, respectively (data not shown).

The CDR sequences of 3A10 mAb according to the Kabat numbering systemare shown in Table 1. The amino acid sequences of light chain variableregion (VL) and heavy chain variable region (VH) of 3A10 mAb are shownin Table 2. The whole light chain and heavy chain sequences of 3A10 mAbare shown in Table 3.

TABLE 1 CDR sequences of 3A10 mAb LCDR1 SGSSSNIGNNYVS (SEQ ID NO: 1)LCDR2 DNNKRPS (SEQ ID NO: 2) LCDR3 GVWDSSLRAEL (SEQ ID NO: 3) HCDR1NAWMS (SEQ ID NO: 6) HCDR2 YISSSGSTIYYADSVKG (SEQ ID NO: 7) HCDR3LAGPYYFDY (SEQ ID NO: 8)

TABLE 2 VL and VH sequences of 3A10 mAb VLQSVVTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQL SEQPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGIDGLRT IDGDEAEYFCGVWDSSLRAELFAGGTKVTVL NO: 4 VHEVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVR SEQQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNS IDLYLQMNSLRAEDTAVYYCARLAGPYYFDYWGQGTLVTV NO: 9 SS

TABLE 3 Light chain and heavy chain sequences of 3A10 mAb LightQSVVTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQL SEQ ID NO: 5  chainPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGIDGLRTGDEAEYFCGVWDSSLRAELFAGGTKVTVLSQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQV THEGSTVEKTVAPTECS HeavyEVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQ SEQ ID NO: 10 chainAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLAGPYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK

Example 3. Binding of the Anti-ENPP3 Monoclonal Antibody to ENPP3

ELISA was performed according to standard protocols, to determinebinding affinity of anti-ENPP3 3A10 mAb to recombinant human ENPP3(AcroBiosystems). Briefly, recombinant human ENPP3 was coated on CorningEIA/RIA high-binding 96-well plates (Corning Inc.) at 100 ng per wellovernight at 4° C. and blocked with 3% nonfat milk in PBS (pH7.4).Five-fold serially diluted antibodies were added and incubated at 37° C.for 1 h. The plates were washed with PBS containing 0.05% TWEEN® 20.Bound antibodies were detected by anti-human IgG(Fc-specific)-peroxidase antibody (Sigma). The assay was developed atroom temperature with TMB substrate (Solarbio) and monitored at 450 nmwith a microplate reader. The half-maximal binding (EC₅₀) was calculatedby fitting the data to the Langmuir adsorption isotherm. The results areshown in FIG. 2 .

The results indicate that 3A10 mAb binds to human ENPP3 with EC₅₀ of107.2 pM, suggesting that 3A10 mAb has high binding affinity to humanENPP3.

Example 4. Binding of the Anti-ENPP3 Monoclonal Antibody to Cancer CellLines

To measure binding ability of the anti-ENPP3 3A10 mAb to cellsurface-associated ENPP3, flow cytometry was carried out using ENPP3positive cancer cell lines SK-Nep-1 and LS174T-ENPP3. About 5×10⁵ cellswere incubated with serial concentrations of antibodies on ice for 1 h,and an IgG4 isotype antibody was used as negative control. The cellswere washed once with PBS containing 0.1% bovine serum albumin (PBSA)and resuspended in 100 μl PBSA. Then 1 μl anti-human IgG(Fc-specific)-FITC conjugate (Sigma) was added and incubated for 30 min.The cells were washed once with PBSA and then used for flow cytometryanalysis. The half-maximal binding (EC₅₀) was calculated by fitting thedata to the Langmuir adsorption isotherm. The results are shown in FIGS.3A and 3B.

The results indicate that 3A10 mAb binds well to SK-Nep-1 andLS174T-ENPP3 cells, suggesting that 3A10 mAb have good binding abilityto ENPP3 positive tumor cell lines.

Example 5. Anti-ENPP3 Monoclonal Antibody Mediated ADCC Killing AgainstHuman Cancer Cell Line

To evaluate ADCC killing of 3A10 mAb, HepG2 cells were used as targetcells and NK cells were used as effector cells. Frozen NK cells wererevived and cultured in RPMI1640 complete medium containing 20% FBS, 1%penicillin/streptomycin, and 50 IU of IL-2 in 5% CO₂ incubator at 37° C.overnight. Target cells HepG2 were prepared to a concentration of1.5×10⁵ cells/mL with the complete medium, and added to a 96-well plateat 100 μL/well, and incubated in a 37° C. incubator supplied with 5% CO₂overnight. At the second day, a serial of concentration (200 μg/mL, 20μg/mL, and 0 μg/mL) of 3A10 mAb were prepared with the complete medium,and an IgG4 isotype antibody was used as negative control. 100 μL ofantibody solutions were added into the 96-well plate containing targetcells. The NK cells were harvested and diluted to 3×10⁵ cells/mL, andthen added to the 96-well plate at 100 μL/well. The plate was incubatedin 5% CO₂ incubator at 37° C. for 48 hours. The final concentrations ofthe antibody are 100 μg/mL, 10 μg/mL, and 0 μg/mL. After 48 hincubation, the culture media was removed and replaced with freshcomplete medium containing 10% CCK8 at 100 μL/well, and the plate wasincubated at 37° C. for 30 minutes. The OD value at 450 nm was measuredwith an ELISA reader. Killing efficiency was calculated according to theequation:(OD _(Tumor+NK+0μg/mL mab)-OD _(Tumor+NK+xμg/mL mab))/OD_(Tumor+NK+0μg/mL mab)×100%,

-   -   in which x represents 10 or 100.

The results of ADCC killing of 3A10 mAb against HepG2 cells is shown inFIG. 4 . The result shows that 3A10 mAb induces around 20% killingagainst tumor cells at 10 μg/mL, and over 36% killing against tumorcells at 100 μg/mL, while the control antibody IgG4 isotype does notinduce any ADCC at the highest concentration of 100 μg/mL, suggestingthat ADCC is triggered by specific binding of 3A10 mAb to ENPP3 positivetumor cells HepG2 and recruiting of NK cells through the Fc portion ofthe mAb. This demonstrates that 3A10 mAb possesses substantialcapability of inducing ADCC killing against ENPP3 positive tumor cells.

Example 6. Construction and Characterization of Anti-ENPP3 BispecificAntibody

Bispecific T cell engager (BITE®) is a novel class of bispecificantibodies that can guide cytotoxic T cells to kill cancer cells bysimultaneously binding to a tumor antigen and a T cell antigen, such asCD3 molecule on T cell surface. HBiTE as described in PCT applicationNo. PCT/US2018/016524 (which is incorporated herein by reference in itsentirety) is a specific form of BiTE®. HBiTE has a light chain and aheavy chain forming a heterodimer. The light chain, from N-terminus toC-terminus, comprises an anti-target (e.g. tumor antigen) VL domain, ananti-CD3 VL-CL and a monomeric human IgG1 Fc (e.g., mFc7.2). The heavychain, from N-terminus to C-terminus, comprises an anti-target VHdomain, an anti-CD3 VH-CH1 and a monomeric human IgG1 Fc (e.g., mFc7.2).Monomeric Fc7.2 contains two amino acid mutations (T366L and Y407H)capable of inhibiting Fc homodimerization. To generate ENPP3×CD3 HBiTE,the VL and VH domains of the above anti-ENPP3 antibody were fused to theN-terminus of VL and VH domains of anti-CD3 Fab via linkers GSGGGGSGGGGS(SEQ ID NO: 21) and GSGGSGGGGSGGGGS (SEQ ID NO: 22), respectively. Theanti-CD3 Fab is further fused to the N terminus of mFc7.2. The lightchain and heavy chain were constructed into a single vector pBYrespectively for expression in mammalian cells. Construction and initialcharacterization of the bispecific antibody targeting ENPP3 and CD3(3A10-based ENPP3×CD3 HBiTE) were performed as follows.

Cloning of the Bispecific Antibody Targeting ENPP3 and CD3

To generate construct of the bispecific antibody 3A10-based ENPP3×CD3HBiTE, following primers were used:

pBY-SP-FP-Not1: (SEQ ID NO: 23)5′GAATGCGGCCGCAAACTACAAGACAGACTTGCAAAAGAAGGCATGCACAGCTCAGCACTGCTCTGTTG 3′ (sense); BI-ENPP3-3A10-VH-FP: (SEQ ID NO: 24)5′TCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCGAGGTGCAGCTGGTGGA 3′ (sense); BI- ENPP3-3A10-VH-RP: (SEQ ID NO: 30)5′ACCTCCGCCTGAACCCCCGGATCCTGAGGAGACGGTGACCAGGGTT 3′ (antisense);BI- ENPP3-3A10-VL-FP: (SEQ ID NO: 26)5′TCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCCAGTCTGTCGTGACGCAGC 3′ (sense); BI- ENPP3-3A10-VL-RP: (SEQ ID NO: 31)5′GCCAGAGCCACCTCCGCCGGATCCTAGGACGGTCACCTTGGTCCCT 3′ (antisense);CD3FC-VH-FP-BamH1: (SEQ ID NO: 32)5′GGATCCGGGGGTTCAGGCGGAGGTGGCTCTGG 3′ (sense); CD3FC-VL-FP:(SEQ ID NO: 33) 5′ GGATCCGGCGGAGGTGGCTCTGGC 3′ (sense); FC-RP-Xba1-DelK:(SEQ ID NO: 34) 5′ TGATCTAGAATTAACCCGGAGACAGGGAGAGGCTCT 3′ (antisense).

For the generation of the bispecific antibody, the gene fragments of VHand VL domains were amplified from 3A10 Fab with primer pairsBI-ENPP3-3A10-VH-FP/BI-ENPP3-3A10-VH-RP andBI-ENPP3-3A10-VL-FP/BI-ENPP3-3A10-VL-RP, respectively. The PCR productswere fused to the 3′ end of a signal peptide by overlapping PCR usingthe primer pairs pBY-SP-FP-Not 1/BI-ENPP3-3A10-VH-RP andpBY-SP-FP-Nott/BI-ENPP3-3A10-VL-RP, respectively. The fragments of ananti-CD3 hSP34 VL-CL and VH-CH1 and a complete Fc were amplified fromthe pBY vector containing the fragments of the CD3 bispecific antibodybased on the same HBiTE format, with primer pairsCD3FC-VL-FP/FC-RP-Xba1-DelK and CD3FC-VH-FP-BamH1/FC-RP-Xba1-DelK,respectively. To obtain the full-length light chain and heavy chain,ENPP3 VL/VH fragments containing signal peptide and CD3 VL-CL-Fc/CD3VH-CH1-Fc were fused by overlapping using primer pairspBY-SP-FP-Not1/Fc-RP-XabI-DelK. The full-length light and heavy chaingene fragment were then cloned into two pBY plasmids via the Not1 andXba1 restriction sites. The 3A10-based ENPP3×CD3 HBiTE was designated asCMD016.

Protein Expression, Purification and Initial Characterization

CMD016 was expressed in 293FS. The plasmids and transfection agent PEIwere mixed at ratio 1:3 and then added into 293FS cell culture. Thecells were continued to grow for 5-7 days after transfection. The cellculture was harvested by centrifugation at 8000 rpm for 20 min. Theculture supernatant containing target proteins were loaded onto GravityEshmunoA Column (Merck), and purified according to the manufacturer'sinstructions.

The purified proteins were subjected to SDS-PAGE. On a non-reducingSDS-PAGE, CMD016 displays an apparent molecular weight (aMW) ofapproximately 120 kDa. On a reducing SDS-PAGE, the heavy chain and lightchain are close to each other with an apparent molecular weight ofapproximately 62 kDa (data not shown). The CDR sequences of CMD016according to the Kabat numbering system are shown in Table 4. The aminoacid sequences of light chain variable region (VL) and heavy chainvariable region (VH) of CMD016 are shown in Table 5. The whole lightchain and heavy chain sequences of CMD016 are shown in Table 6.

TABLE 4 CDR sequences of CMD016 LCDR1 against ENPP3SGSSSNIGNNYVS (SEQ ID NO: 1) LCDR2 against ENPP3 DNNKRPS (SEQ ID NO: 2)LCDR3 against ENPP3 GVWDSSLRAEL (SEQ ID NO: 3) HCDR1 against ENPP3NAWMS (SEQ ID NO: 6) HCDR2 against ENPP3 YISSSGSTIYYADSVKG (SEQ IDNO: 7) HCDR3 against ENPP3 LAGPYYFDY (SEQ ID NO: 8) LCDR1 against CD3RSSTGAVTTSNYAN (SEQ ID NO: 11) LCDR2 against CD3 GANKRAP (SEQ ID NO: 12)LCDR3 against CD3 ALWYSNLWV (SEQ ID NO: 13) HCDR1 against CD3GFTFNTY (SEQ ID NO: 16) HCDR2 against CD3 RSKYNNYA (SEQ ID NO: 17)HCDR3 against CD3 HGNFGSSYVSYFAY (SEQ ID NO: 18)

TABLE 5 VL and VH sequences of CMD016 VL againstQSVVTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWY SEQ ID NO: 4 ENPP3QQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGIDGLRTGDEAEYFCGVWDSSLRAELFAGGTKVTVL VL againstEIVVTQSPATLSVSPGERATLSCRSSTGAVTTSNYAN SEQ ID NO: 14 CD3WVQQKPGQAPRGLIGGANKRAPGVPARFSGSLSGDEATLTISSLQSEDFAVYYCALWYSNLWVFGQGTKLEIK VH againstEVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSW SEQ ID NO: 9 ENPP3VRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLAGPYYFDYWG QGTLVTVSS VH againstEVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNW SEQ ID NO: 19 CD3VRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGSSY VSYFAYWGQGTTVTVSS

TABLE 6 Light chain and heavy chain sequences of CMD016 Light chainQSVVTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWY SEQ ID NO: 15QQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGIDGLRTGDEAEYFCGVWDSSLRAELFAGGTKVTVLGSGGGGSGGGGSEIVVTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGANKRAPGVPARFSGSLSGDEATLTISSLQSEDFAVYYCALWYSNLWVFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLHSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG Heavy chainEVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMS SEQ ID NO: 20WVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISR DNAKNSLYLQMNSLRAEDTAVYYCARLAGPYYFDYWGQGTLVTVSSGSGGSGGGGSGGGGSEVQLVESG GGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSK NTLYLQMNSLRAEDTAVYYCARHGNFGSSYVSYFAYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLHSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Example 7. Binding of the Bispecific Antibody to ENPP3 and CD3

To determine binding affinity of the bispecific antibody CMD016 to bothENPP3 and CD3, ELISA experiments were performed as described in Example3, with the coating proteins of human ENPP3 or human CD3. The resultsare shown in FIGS. 5A-5B.

The results indicate that CMD016 binds to human ENPP3 with EC₅₀ of 2.29nM (FIG. 5A), and binds to human CD3 with EC₅₀ of 5.28 nM (FIG. 5B).These results suggest that CMD016 can bind to both ENPP3 and CD3 withhigh affinity.

Example 8. Binding of the Bispecific Antibody to Cancer Cell Lines

To determine binding affinity of the bispecific antibody CMD016 to cellsurface-associated ENPP3 and CD3, flow cytometry was carried out usingENPP3 positive cancer cell line SK-Nep-1 and CD3 positive Jurkat cellline. The procedures were similar to those described in Example 4. Theresults were shown in FIGS. 6A-6B.

The results indicate that CMD016 binds well to SK-Nep-1 and Jurkatcells. This suggests that CMD016 can bind to both cancer cellsexpressing ENPP3 and cells expressing CD3.

Example 9. Bispecific Antibody Mediated Killing of Human Cancer CellLines

Bispecific T cell engager can simultaneously bind to a tumor antigen anda T cell antigen (e.g., CD3 molecular on T cell surface) causingaggregation and activation of T cells, eventually leading to the killingof tumor cells. To evaluate killing efficiency of the bispecificantibody CMD016, CCK8 assay was performed using tumor cell lineLS174T-ENPP3 stably expressing ENPP3 as target cells. ENPP3 negativecell line LS174T was used as negative control.

3×10⁴ LS174T-ENPP3 cells were seeded in 100 μl RPMI 1640 complete mediumovernight. Meanwhile, frozen PBMCs were revived and inoculated in 30 mLRPMI 1640 complete medium overnight. At the second day, 1.5×10⁵ PBMCs in50 μl RPMI 1640 complete medium were added. Then, 50 μl antibodies(5-fold serially diluted from 4 μg/ml) were added into each well. 48 hafter incubation, the medium was removed and 100 μl RPMI 1640 completemedium containing 10% CCK8 was added and incubated 30 minutes in a CO₂incubator. Cell killing activity was measured by using microplate readeraccording to the manufacturer's instructions.

The results were shown in FIG. 7 . The results indicate that nearly 80%LS174T-ENPP3 cells were killed in the presence of CMD016 and PBMCs. TheEC₅₀ of LS174T-ENPP3 killing by CMD016 is 10.98 ng/ml. The results havedemonstrated CMD016 possesses potent killing capability againstLS174T-ENPP3 cells stably expressing ENPP3, supporting its anti-tumorefficacy.

Example 10. Bispecific Antibody Mediated Inhibition of Tumor Growth inMice Pharmacokinetic Measurement

Three NOD/SCID mice were administered intravenously with 300 μg CMD016on day 0. Plasma samples were collected at time points 24 hr, 48 hr, 72hr and 96 hr after treatment and used for measurement of antibody serumconcentration by ELISA.

The result indicated that the serum concentration of CMD016 wasgradually decreased but still maintained a relatively high level untilthe end point, and the calculated serum half-life (t_(1/2)) was around115.39 hours (FIG. 8 ).

In-Vivo Tumor Growth Inhibition

2.5×10⁶ effector cells human PBMCs and 2.5×10⁶ ENPP3 expressing tumorcells LS174T-ENPP3 were mixed and inoculated subcutaneously into theright side of abdomen of B-NDG mice. In experiment groups, 33.3 μg/kg(low-dose group), 100 μg/kg (medium-dose group) and 300 μg/kg (high-dosegroup) of CMD016 were injected intravenously into mice, respectively.These mice were dosed twice a week. The negative control group mice weredosed intravenously with PBS. Tumor volume and body weight of mice weremeasured at day 0, 3, 6, 10, 13 and 21 after the treatment. After threeweeks of the treatment, mice were sacrificed and tumor weight wasmeasured. Tumor growth inhibition rate was calculated by using thefollowing formula: (Average tumor weight of control group—average tumorweight of experiment group)/average tumor weight of control group.

The results indicate that CMD016 exhibits potent inhibition of tumorgrowth in all of the low-dose group, medium-dose group and high-dosegroup (FIG. 9A). The body weight of mice in all groups only has minorvariation (FIG. 9B). All of the low-dose group, medium-dose group andhigh-dose group show over 95% inhibition rate of tumor growth (FIG. 9C),suggesting significant in vivo anti-tumor effect of CMD016.

In summary, the results have demonstrated that CMD016 has a long serumhalf-life, and can specifically and potently inhibit growth of the tumorcells expressing ENPP3, suggesting its potential for treating ENPP3positive cancers.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments described herein may beemployed. It is intended that the following claims define the scope ofthe invention and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

The invention claimed is:
 1. A method of treating a cancer in a humansubject having the cancer, comprising administering to the human subjectan effective amount of an antibody molecule that specifically binds toENPP3 or an antigen binding fragment thereof comprising a light chainvariable region (VL) and a heavy chain variable region (VH), wherein theVL comprises LCDRs 1-3 having the amino acid sequences as set forth inSEQ ID NOs: 1-3 respectively, and the VH comprises HCDRs 1-3 having theamino acid sequences as set forth in SEQ ID NOs: 6-8 respectively, andwherein the cancer is an ENPP3 positive cancer.
 2. The method accordingto claim 1, wherein the VL comprises the amino acid sequence as setforth in SEQ ID NO: 4 or an amino acid sequence having at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 4, and the VH comprises the amino acidsequence as set forth in SEQ ID NO: 9 or an amino acid sequence havingat least 80%, at least 85%, at least 90%, at least 95%, at least 98%, orat least 99% sequence identity to SEQ ID NO:
 9. 3. The method accordingto claim 1, wherein the antibody molecule is of an isotype selected fromthe group consisting of IgG, IgA, IgM, IgE and IgD.
 4. The methodaccording to claim 1, wherein the antibody molecule is of a subtypeselected from the group consisting of IgG1, IgG2, IgG3, and IgG4.
 5. Themethod according to claim 1, wherein the antigen binding fragment isselected from the group consisting of Fab, Fab′, F(ab′)₂, Fv, scFv, andds-scFv.
 6. The method according to claim 1, wherein the antibodymolecule comprises a light chain comprising an amino acid sequence asset forth in SEQ ID NO: 5 or an amino acid sequence having at least 80%,at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 5 and a heavy chain comprising an aminoacid sequence as set forth in SEQ ID NO: 10 or an amino acid sequencehaving at least 80%, at least 85%, at least 90%, at least 95%, at least98%, or at least 99% sequence identity to SEQ ID NO:
 10. 7. The methodaccording to claim 1, wherein the antibody molecule is a bispecificantibody molecule which further comprises a second antigen bindingregion that binds to a second antigen, wherein the second antigen is atumor associated antigen, an immune cell antigen, or a T-cell antigen.8. The method according to claim 7, wherein the T-cell antigen isselected from the group consisting of T cell receptor (TCR), CD3, CD4,CD8, CD16, CD25, CD28, CD38, CD44, CD62L, CD69, ICOS, 41-BB (CD137), andNKG2D.
 9. The method according to claim 7, wherein the second antigen isCD3, and the second antigen binding region comprises a VL comprising theamino acid sequence as set forth in SEQ ID NO: 14 and a VH comprisingthe amino acid sequence as set forth in SEQ ID NO:
 19. 10. The methodaccording to claim 9, wherein the VL of the second antigen bindingregion is linked to the C-terminus of the VL of the antibody moleculethat specifically binds to ENPP3, optionally via a first linker, and theVH of the second antigen binding region is linked to the C-terminus ofthe VH of the antibody molecule that specifically binds to ENPP3,optionally via a second linker, wherein the first linker and the secondlinker are the same or different.
 11. The method according to claim 10,wherein the first linker comprises the amino acid sequence as set forthin SEQ ID NO: 21, and the second linker comprises the amino acidsequence as set forth in SEQ ID NO:
 22. 12. The method according toclaim 9, wherein the bispecific antibody molecule comprises a lightchain comprising the amino acid sequence as set forth in SEQ ID NO: 15or an amino acid sequence having at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% sequence identity toSEQ ID NO: 15 and a heavy chain comprising the amino acid sequence asset forth in SEQ ID NO: 20 or an amino acid sequence having at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99% sequence identity to SEQ ID NO:
 20. 13. The method according toclaim 7, wherein the bispecific antibody molecule is a bispecific T-cellengager.
 14. The method according to claim 1, wherein the cancer isselected from the group consisting of mastocytosis, leukemia, kidneycancer, lung cancer, gastric cancer, ovarian cancer, breast cancer,pancreatic cancer, colon cancer, colorectal cancer, bile duct cancer,liver cancer, and Wilms tumor.
 15. The method according to claim 1,further comprising administering to the human subject a secondtherapeutic agent.
 16. The method according to claim 15, wherein thesecond therapeutic agent is selected from an antibody, achemotherapeutic agent and a small molecule drug.
 17. The methodaccording to claim 15, wherein the second therapeutic agent is selectedfrom a Bruton's tyrosine kinase (BTK) inhibitor, a PI3K inhibitor, aHDAC inhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1/PD-L1inhibitor, a LAG3 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, aTIM3 inhibitor, and glucocorticoid.
 18. The method according to claim 1,wherein the VL comprises the amino acid sequence as set forth in SEQ IDNO: 4, and the VH comprises the amino acid sequence as set forth in SEQID NO:
 9. 19. The method according to claim 1, wherein the antibodymolecule comprises a light chain comprising the amino acid sequence asset forth in as set forth in SEQ ID NO: 5 and a heavy chain comprisingthe amino acid sequence as set forth in as set forth in SEQ ID NO: 10.20. The method according to claim 9, wherein the bispecific antibodymolecule comprises a light chain comprising the amino acid sequence asset forth in as set forth in SEQ ID NO: 15 and a heavy chain comprisingthe amino acid sequence as set forth in as set forth in SEQ ID NO: 20.